U.S. patent application number 16/619388 was filed with the patent office on 2020-09-03 for method for synthesizing peptide containing n-substituted amino acid.
The applicant listed for this patent is Chugai Seiyaku Kabushiki Kaisha. Invention is credited to Takashi EMURA, Terushige MURAOKA, Kenichi NOMURA, Mikimasa TANADA.
Application Number | 20200277327 16/619388 |
Document ID | / |
Family ID | 1000004868536 |
Filed Date | 2020-09-03 |
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United States Patent
Application |
20200277327 |
Kind Code |
A1 |
NOMURA; Kenichi ; et
al. |
September 3, 2020 |
METHOD FOR SYNTHESIZING PEPTIDE CONTAINING N-SUBSTITUTED AMINO
ACID
Abstract
Methods of producing a peptide containing an N-substituted amino
acid or N-substituted amino acid analog of the present invention
include the steps of: preparing an Fmoc-protected amino acid, an
Fmoc-protected amino acid analog, or an Fmoc-protected peptide;
deprotecting a protecting group which have an Fmoc skeleton of the
Fmoc-protected amino acid and such by using a base; and forming an
amide bond by adding a new Fmoc-protected amino acid and such; and
when the peptide is produced by a solid-phase method, the obtained
peptide is cleaved off from the solid phase under conditions of
weaker acidity than TFA. Furthermore, at least one side chain of
the obtained peptide has a protecting group that is not deprotected
under basic conditions and is deprotected under conditions of
weaker acidity than TFA.
Inventors: |
NOMURA; Kenichi; (Shizuoka,
JP) ; MURAOKA; Terushige; (Shizuoka, JP) ;
TANADA; Mikimasa; (Shizuoka, JP) ; EMURA;
Takashi; (Shizuoka, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chugai Seiyaku Kabushiki Kaisha |
Tokyo |
|
JP |
|
|
Family ID: |
1000004868536 |
Appl. No.: |
16/619388 |
Filed: |
June 8, 2018 |
PCT Filed: |
June 8, 2018 |
PCT NO: |
PCT/JP2018/021998 |
371 Date: |
December 4, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 11/02 20130101;
C07K 1/061 20130101 |
International
Class: |
C07K 1/06 20060101
C07K001/06; C07K 11/02 20060101 C07K011/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 9, 2017 |
JP |
2017-114073 |
Claims
1. A method of producing a peptide comprising at least one
N-substituted amino acid or N-substituted amino acid analog,
wherein the method comprises the steps of: 1) preparing an amino
acid (Fmoc-protected amino acid) comprising at least one each of
following functional groups i) and ii), an amino acid analog
(Fmoc-protected amino acid analog) comprising at least one each of
following i) and ii), or a peptide (Fmoc-protected peptide)
comprising either one or both of the Fmoc-protected amino acid and
the Fmoc-protected amino acid analog: i) a main chain amino group
protected by at least one protecting group having an Fmoc skeleton;
and ii) at least one free carboxylic acid group or active
esterified carboxylic acid group; 2) making the Fmoc-protected
amino acid, the Fmoc-protected amino acid analog, or the
Fmoc-protected peptide prepared in step 1) to be supported onto a
solid phase; 3) deprotecting the protecting group having the Fmoc
skeleton of the Fmoc-protected amino acid, the Fmoc-protected amino
acid analog, or the Fmoc-protected peptide, which is supported onto
the solid phase, by using a base to expose its amino group; 4)
forming an amide bond by adding a new Fmoc-protected amino acid, a
new Fmoc-protected amino acid analog, or a new Fmoc-protected
peptide; and 5) cleaving the peptide obtained in step 4) off from
the solid phase under a condition of weaker acidity than TFA.
2. The production method of claim 1, wherein at least one side
chain of the amino acid or amino acid analog constituting the
peptide obtained in step 4) is protected by a protecting group that
is not deprotected under a basic condition but is deprotected by a
first acid, and wherein the method further comprises before or
after step 5), a step of deprotecting the protecting group using
the first acid; and wherein in step 5), the peptide is cleaved off
using a second acid, and wherein the first acid and the second acid
are both weaker acids than TFA and the acidity of the first acid is
higher than the acidity of the second acid.
3. A method of producing a peptide comprising at least one
N-substituted amino acid or N-substituted amino acid analog,
wherein the method comprises the steps of: 1) preparing an amino
acid (Fmoc-protected amino acid) comprising at least one each of
following functional groups i) and ii), an amino acid analog
(Fmoc-protected amino acid analog) comprising at least one each of
following functional groups i) and ii), or a peptide
(Fmoc-protected peptide) comprising either one or both of the
Fmoc-protected amino acid and the Fmoc-protected amino acid analog:
i) a main chain amino group protected by at least one protecting
group having an Fmoc skeleton; and ii) at least one free carboxylic
acid group or active esterified carboxylic acid group; 2)
deprotecting the protecting group having the Fmoc-skeleton of the
Fmoc-protected amino acid, the Fmoc-protected amino acid analog, or
the Fmoc-protected peptide, by using a base to expose its amino
group; 3) forming an amide bond by adding a new Fmoc-protected
amino acid, a new Fmoc-protected amino acid analog, or a new
Fmoc-protected peptide, wherein at least one side chain of the
amino acid or amino acid analog constituting a peptide obtained in
this step has a protecting group that is not deprotected under a
basic condition and is deprotected under a condition having weaker
acidity than TFA; and 4) deprotecting the protecting group of the
side chain under the condition having weaker acidity than TFA.
4. The production method of claim 3, wherein peptide production is
carried out by a solid phase method.
5. The production method of claim 4, which further comprises before
or after step 4), a step of cleaving the peptide obtained in step
3) off from the solid phase under a condition further weaker than
the weakly-acidic condition used in step 4).
6. The production method of claim 3, wherein peptide production is
carried out by a liquid phase method.
7. The production method of claim 1, wherein step 4) further
comprises the steps of: deprotecting the protecting group having
the Fmoc skeleton on the newly added Fmoc-protected amino acid, the
newly added Fmoc-protected amino acid analog, or the newly added
Fmoc-protected peptide, by using a base to expose its amino group;
and forming an amide bond by further adding a new Fmoc-protected
amino acid, a new Fmoc-protected amino acid analog, or a new
Fmoc-protected peptide, and wherein these steps are repeated once
or multiple times.
8. The production method of claim 1, wherein the produced peptide
comprises on its C-terminal side an amino acid residue or an amino
acid analogue residue comprising one reactive site, and comprises
on its N-terminal side an amino acid residue, an amino acid
analogue residue, or a carboxylic acid analog comprising the other
reactive site.
9. The production method of claim 8, which further comprises the
step of bonding said reactive site and said other reactive site to
cyclize the peptide.
10. The production method of claim 9, wherein the amino acid
residue, the amino acid analogue residue, or the carboxylic acid
analog having said other reactive site is at the N terminus and the
bonding is an amide bonding or a carbon-carbon bonding.
11. (canceled)
12. The production method of claim 1, wherein the step performed
under a condition having weaker acidity than TFA is performed using
a weakly acidic solution comprising a weak acid having an aqueous
pKa value of 0 to 9 in a solvent having an aqueous pKa value of 5
to 14 and whose ionization ability value Y.sub.OTs is positive.
13. The production method of claim 12, wherein the solvent is
fluoroalcohol.
14. The production method of claim 13, wherein the fluoroalcohol is
TFE or HFIP.
15. The production method of claim 2, wherein the side chain
protecting group is a protecting group which is deprotected in the
range of pH 1 to pH 7, or a protecting group which is deprotected
in 10% or lower concentration of TFA.
16. The production method of claim 2, wherein the side chain
protecting group is selected from following a) to d): a) when the
side chain protecting group is a protecting group for the side
chain hydroxyl group of Ser, Thr, Hyp, and derivatives thereof, any
one protecting group selected from a MOM skeleton, a Bn skeleton, a
Dpm skeleton, a Trt skeleton, a silyl skeleton, and a Boc skeleton
represented by the general formulae below; b) when the side chain
protecting group is a protecting group for the side chain hydroxyl
group of Tyr and derivatives thereof, any one protecting group
selected from a MOM skeleton, a Bn skeleton, a Dpm skeleton, a Trt
skeleton, a silyl skeleton, a Boc skeleton, and a tBu skeleton
represented by the general formulae below; c) when the side chain
protecting group is a protecting group for the side chain imidazole
ring of His and derivatives thereof, any one protecting group
selected from a MOM skeleton, a Bn skeleton, and a Trt skeleton
represented by the general formulae below; and d) when the side
chain protecting group is a protecting group for the side chain
carboxylic acid group of Asp, Glu, and derivatives thereof, any one
protecting group selected from a MOM skeleton, a Bn skeleton, a Dpm
skeleton, a Trt skeleton, a tBu skeleton, a phenyl-EDOTn skeleton,
which are represented by the following general formulae, and an
orthoester skeleton in which a carbon atom of the carboxylic acid
group to be protected is substituted with three alkoxy groups:
<a protecting group having a MOM skeleton> ##STR00138##
(wherein R1 is H, R2 is H, and X is methyl, benzyl,
4-methoxybenzyl, 2,4-dimethoxybenzyl, 3,4-dimethoxybenzyl, or
2-trimethylsilylethyl; R1 is methyl, R2 is H, and X is ethyl; R1,
R2, and R3 are all methyl; or R1 and X together form
--CH.sub.2--CH.sub.2--CH.sub.2-- or
--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--, and R2 is H, wherein
when any one of R1, R2, and X is methyl or ethyl, these groups may
further be substituted with alkyl, benzyl, or aryl); <a
protecting group having a Bn skeleton> ##STR00139## (wherein R1
to R5 are each independently H, alkyl, aryl, or halogen, and R6 and
R7 are alkyl; R1, R2, R4, and R5 are each independently H, alkyl,
aryl, or halogen, R3 is methoxy, and R6 and R7 are H; R1 and R3 are
methoxy, R2, R4, and R5 are each independently H, alkyl, aryl, or
halogen, and R6 and R7 are H; or R1, R4, and R5 are each
independently H, alkyl, aryl, or halogen, and R2 and R3 together
form --O--CH2-O--); <a protecting group having a Dpm
skeleton> ##STR00140## (wherein R1 to R10 are each independently
H, alkyl, aryl, alkoxy, or halogen; or R1 to R4 and R7 to R10 are
each independently H, alkyl, aryl, alkoxy, or halogen, and R5 and
R6 together form --O-- or --CH2-CH2-); <a protecting group
having a Trt skeleton> ##STR00141## (wherein R1 to R15 are each
independently H, alkyl, aryl, alkoxy, or halogen; R1, R2, and R4 to
R15 are each independently H, alkyl, aryl, alkoxy, or halogen, and
R3 is methyl or methoxy; R1 is Cl, and R2 to R15 are each
independently H, alkyl, aryl, alkoxy, or halogen; or R1 to R4 and
R7 to R15 are each independently H, alkyl, aryl, alkoxy, or
halogen, and R5 and R6 together form --O--); <a protecting group
having a silyl skeleton> ##STR00142## (wherein R1 to R3 are each
independently alkyl or aryl); <a protecting group having a Boc
skeleton> ##STR00143## (wherein R1 to R9 are each independently
H, alkyl, or aryl); <a protecting group having a tBu
skeleton> ##STR00144## (wherein R1 to R9 are each independently
H, alkyl, or aryl); and <a protecting group having a
phenyl-EDOTn skeleton> ##STR00145## (wherein R1 to R3 are each
independently H or methoxy).
17. The production method of claim 3, wherein step 3) further
comprises the steps of: deprotecting the protecting group having
the Fmoc skeleton on the newly added Fmoc-protected amino acid, the
newly added Fmoc-protected amino acid analog, or the newly added
Fmoc-protected peptide, by using a base to expose its amino group;
and forming an amide bond by further adding a new Fmoc-protected
amino acid, a new Fmoc-protected amino acid analog, or a new
Fmoc-protected peptide, and wherein these steps are repeated once
or multiple times.
18. The production method of claim 3, wherein the produced peptide
comprises on its C-terminal side an amino acid residue or an amino
acid analogue residue comprising one reactive site, and comprises
on its N-terminal side an amino acid residue, an amino acid
analogue residue, or a carboxylic acid analog comprising the other
reactive site.
19. The production method of claim 18, which further comprises the
step of bonding said reactive site and said other reactive site to
cyclize the peptide.
20. The production method of claim 19, wherein the amino acid
residue, the amino acid analogue residue, or the carboxylic acid
analog having said other reactive site is at the N terminus and the
bonding is an amide bonding or a carbon-carbon bonding.
21. The production method of claim 3, wherein the step performed
under a condition having weaker acidity than TFA is performed using
a weakly acidic solution comprising a weak acid having an aqueous
pKa value of 0 to 9 in a solvent having an aqueous pKa value of 5
to 14 and whose ionization ability value Y.sub.OTs is positive.
22. The production method of claim 21, wherein the solvent is
fluoroalcohol.
23. The production method of claim 22, wherein the fluoroalcohol is
TFE or HFIP.
24. The production method of claim 3, wherein the side chain
protecting group is a protecting group which is deprotected in the
range of pH 1 to pH 7, or a protecting group which is deprotected
in 10% or lower concentration of TFA.
25. The production method of claim 3, wherein the side chain
protecting group is selected from following a) to d): a) when the
side chain protecting group is a protecting group for the side
chain hydroxyl group of Ser, Thr, Hyp, and derivatives thereof, any
one protecting group selected from a MOM skeleton, a Bn skeleton, a
Dpm skeleton, a Trt skeleton, a silyl skeleton, and a Boc skeleton
represented by the general formulae below; b) when the side chain
protecting group is a protecting group for the side chain hydroxyl
group of Tyr and derivatives thereof, any one protecting group
selected from a MOM skeleton, a Bn skeleton, a Dpm skeleton, a Trt
skeleton, a silyl skeleton, a Boc skeleton, and a tBu skeleton
represented by the general formulae below; c) when the side chain
protecting group is a protecting group for the side chain imidazole
ring of His and derivatives thereof, any one protecting group
selected from a MOM skeleton, a Bn skeleton, and a Trt skeleton
represented by the general formulae below; and d) when the side
chain protecting group is a protecting group for the side chain
carboxylic acid group of Asp, Glu, and derivatives thereof, any one
protecting group selected from a MOM skeleton, a Bn skeleton, a Dpm
skeleton, a Trt skeleton, a tBu skeleton, a phenyl-EDOTn skeleton,
which are represented by the following general formulae, and an
orthoester skeleton in which a carbon atom of the carboxylic acid
group to be protected is substituted with three alkoxy groups:
<a protecting group having a MOM skeleton> ##STR00146##
(wherein R1 is H, R2 is H, and X is methyl, benzyl,
4-methoxybenzyl, 2,4-dimethoxybenzyl, 3,4-dimethoxybenzyl, or
2-trimethylsilylethyl; R1 is methyl, R2 is H, and X is ethyl; R1,
R2, and R3 are all methyl; or R1 and X together form
--CH.sub.2--CH.sub.2--CH.sub.2-- or
--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--, and R2 is H, wherein
when any one of R1, R2, and X is methyl or ethyl, these groups may
further be substituted with alkyl, benzyl, or aryl); <a
protecting group having a Bn skeleton> ##STR00147## (wherein R1
to R5 are each independently H, alkyl, aryl, or halogen, and R6 and
R7 are alkyl; R1, R2, R4, and R5 are each independently H, alkyl,
aryl, or halogen, R3 is methoxy, and R6 and R7 are H; R1 and R3 are
methoxy, R2, R4, and R5 are each independently H, alkyl, aryl, or
halogen, and R6 and R7 are H; or R1, R4, and R5 are each
independently H, alkyl, aryl, or halogen, and R2 and R3 together
form --O--CH2--O--); <a protecting group having a Dpm
skeleton> ##STR00148## (wherein R1 to R10 are each independently
H, alkyl, aryl, alkoxy, or halogen; or R1 to R4 and R7 to R10 are
each independently H, alkyl, aryl, alkoxy, or halogen, and R5 and
R6 together form --O-- or --CH2-CH2-); <a protecting group
having a Trt skeleton> ##STR00149## (wherein R1 to R15 are each
independently H, alkyl, aryl, alkoxy, or halogen; R1, R2, and R4 to
R15 are each independently H, alkyl, aryl, alkoxy, or halogen, and
R3 is methyl or methoxy; R1 is Cl, and R2 to R15 are each
independently H, alkyl, aryl, alkoxy, or halogen; or R1 to R4 and
R7 to R15 are each independently H, alkyl, aryl, alkoxy, or
halogen, and R5 and R6 together form --O--); <a protecting group
having a silyl skeleton> ##STR00150## (wherein R1 to R3 are each
independently alkyl or aryl); <a protecting group having a Boc
skeleton> ##STR00151## (wherein R1 to R9 are each independently
H, alkyl, or aryl); <a protecting group having a tBu
skeleton> ##STR00152## (wherein R1 to R9 are each independently
H, alkyl, or aryl); and <a protecting group having a
phenyl-EDOTn skeleton> ##STR00153## (wherein R1 to R3 are each
independently H or methoxy).
Description
TECHNICAL FIELD
[0001] The present invention relates to novel methods for
synthesizing peptides which allow for the synthesis with high
purity and high synthesis efficiency, in synthesizing peptides
comprising N-substituted amino acids.
BACKGROUND ART
[0002] Peptides are highly valuable chemical species, and 40 or
more types of peptides have been placed on the market as
pharmaceuticals (NPL 1). Among them, for cyclic peptides and
N-methylated (or N-alkylated) unnatural peptides, improvement of
membrane permeability for the improved lipophilicity and
improvement of metabolic stability for the acquisition of
resistance to hydrolytic enzymes (hydrolases) have been anticipated
(NPL 2). Recently, investigation on cyclic peptides that are
drug-like (drug-likeness: preferably, it indicates that both
membrane permeable and metabolically stable characteristics are
achieved), the drug-likeness being the key to accomplish transfer
into cells and to allow formulation as oral agents, is progressing
(NPLs 3 and 4). Furthermore, a patent document elucidating the
conditions necessary for drug-like cyclic peptides was published
(PTL 1), and the importance and awareness of such peptides in drug
development have been increasing.
[0003] On the other hand, progresses in the development of methods
of synthesizing peptides comprising many unnatural amino acids such
as those represented by N-alkyl amino acids are relatively narrow.
In most cases, techniques established for natural peptides have
been directly applied to unnatural peptides.
[0004] The Fmoc method and the Boc method are widely known as
peptide synthesis methods, and most of the findings regarding these
methods have been obtained from the development of methods for
synthesizing natural peptides. An Fmoc group is stable to acids;
therefore, when an N-terminal amino group is protected by an Fmoc
group, its deprotection reaction is performed using bases such as
DBU and piperidine. Thus, for example, protecting groups that can
be deprotected (deprotectable) by acids are used as protecting
groups for peptide side chain functional groups, and peptide chain
is elongated by selectively deprotecting N-terminal amino group. As
protecting groups often used, t-butyl (tBu), trityl (Trt), and such
groups deprotectable by acids at the level of trifluoroacetic acid
(TFA) can be employed for protecting amino acid side chains in the
Fmoc method, and the step of cleaving the peptide from the resin
and deprotection of protecting groups of the side chain functional
groups can be performed under milder conditions compared to the Boc
method.
[0005] However, even in a solid-phase synthesis method by the Fmoc
method, which allows cleavage from resins and deprotection of
protecting groups of side-chain functional groups under relatively
mild conditions, the following problems have been revealed in the
synthesis of N-alkylated peptides during the step of cleavage from
resins or deprotection of protecting groups on the side-chain
functional groups using TFA.
[0006] When synthesizing peptides by the usual Fmoc method, it is
common to use TFA for the step of cleavage from the resin and
deprotection of protecting groups of side-chain functional groups.
In most cases, cleavage reaction from the resin and deprotection
reaction of the side-chain functional groups are performed
simultaneously using an aqueous 90% TFA solution. However, it has
been known that in the case of N-methylated peptides, particularly
peptides with sequences having consecutive N-methyl amino acids, a
side reaction occurs where acid hydrolysis via oxazolonium proceeds
and the peptide chain is cleaved (NPLs 5 and 6). Furthermore, it
has been known that in the case of peptides comprising in their
sequences amino acids having an .quadrature.-hydroxy group such as
serine and threonine, N- to O-acyl shift reaction can also proceed
as a side reaction in addition to the acid hydrolysis in these
steps that use TFA, which results in the depsipeptide formation
(NPLs 7 and 8).
[0007] Measures to avoid this problem of hydrolysis in the cleavage
step and deprotection step using acids are being taken, such as
using a low-concentration TFA solution and controlling the reaction
time to be short. For example, according to the report by Albericio
et al., in the solid-phase synthesis of a peptide named
NMe-IB-01212, peptide degradation at the N-Me site was observed
when deprotection of Boc group on an amino group included in an
N-methylated cyclic hexapeptide is carried out in a TFA-DCM (1:1)
solution. Although attempts for improvement have been made to avoid
degradation by using lower concentrations of TFA and reducing the
reaction time to a minimum, sufficient improvement has not been
attained (NPL 9). in the first place, protecting groups widely used
in conventional peptide synthesis have shown cases where the
deprotection step using a low-concentration TFA solution results in
deprotection reaction on the side chains proceeding extremely
slowly or not proceeding whereas cleavage reaction of peptides from
the resin proceeding at a satisfactory speed.
[0008] Furthermore, to prevent cleavage of Ac-MePhe at the N
terminus which proceeds via the same reaction mechanism as
hydrolysis of highly N-methylated peptides, Fang et al. used TFA
and decreased the reaction temperature to 4.degree. C. to deprotect
the Pbf group, which is a protecting group for the Arg side chain
(NPL 10). However, even by using this method which decreases the
temperature, complete prevention of Ac-MePhe cleavage was
difficult, and the method could just stop the reaction when the
generated level of the desired product reaches its maximum.
[0009] In addition to the problem during deprotection, the problem
of low reactivity in the elongation step is also known. When an
N-methyl amino acid comes to the N terminus of the amide bond which
is newly formed, the amide formation reaction (elongation reaction)
with a subsequent amino acid may not proceeds sufficiently because
of the bulkiness of its secondary amine (NPLs 2 and 5).
[0010] For this problem in the elongation step, measures to
decrease unreacted site by repeating identical reaction condition
twice or more times have been taken (the method repeating twice is
called double coupling). Furthermore, regarding activation of the
amino acid to be condensed, effort has been made to improve
condensation efficiency, for example, by changing to highly active
acid halides (NPL 11). However, repeating the same reaction
condition as in the double coupling will double or more the time
and reagent cost spent; and performing the condensation using acid
halides will require preparation of the acid halides at the time of
use, and it also adds an unattended concern of whether the
generated acid halides can exist stably during the peptide
synthesis step. Furthermore, generation of HCl and HF by the
reaction invites a possibly problematic concern that deprotection
reaction may unintendedly proceed.
[0011] Other measures for improving low reactivity in the
elongation step have been tried such as increasing the condensation
efficiency by decreasing the amount loaded onto the resin to reduce
the density of the peptide chains on the solid phase and by
increasing the concentration of the reaction solution (NPL
Recently, there have been efforts to improve the condensation
efficiency by increasing the reaction temperature through
irradiation of microwaves (NPLs 12 and 13).
[0012] However, in N-methylated peptide syntheses, there are no
reports of radical solutions for concerns for the decreased purity
and yield of the peptides to be synthesized, and that the desired
product will not be obtained at all in some cases.
PRIOR ART REFERENCES
Patent Literature
[0013] [PTL 1] WO 2013/100132 A1
Non-Patent Literature
[0013] [0014] [NPL 1] S. R. Gracia, et al., Synthesis of chemically
modified bioactive peptides: recent advances, challenges and
developments for medicinal chemistry. Future Med. Chem., 2009, 1,
1289. [0015] [NPL 2] J. Chatterjee, et al., N-Methylation of
peptides: A new perspective in medicinal chemistry. Acc. Chem.
Res., 2008, 41, 1331. [0016] [NPL 3] J. E. Bock, et al., Getting in
Shape: Controlling Peptide Bioactivity and Bioavailability Using
Conformational Constraints, ACS Chem. Biol., 2013, 8, 488. [0017]
[NPL 4] K. Jpsephson, et al., mRNA display: from basic principles
to macrocycle drug discovery. Drug Discovery Today, DOI:
10.1016/j.drudis.2013. 10.011 [0018] [NPL 5] M. Teixido, et al.,
Solid-phase synthesis and characterization of N-methyl-rich
peptides. J. Peptide Res., 2005, 65, 153. [0019] [NPL 6] J. Urban,
et al., Lability of N-alkylated peptides towards TFA cleavage. Int.
J. Pept. Prot. Res., 1996, 47, 182. [0020] [NPL 7] L. A. Carpino,
et al., Dramatically enhanced N.fwdarw.O acyl migration during the
trifluoroacetic acid-based deprotection step in solid phase peptide
synthesis. Tetrahedron Lett., 2005, 46, 1361. [0021] [NPL 8] H.
Eberhard, et al. N.fwdarw.O-Acyl shift in Fmoc-based synthesis of
phosphopeptides. Org. Biomol. Chem., 2008, 6, 1349. [0022] [NPL 9]
E. Marcucci, et al., Solid-Phase Synthesis of NMe-IB-01212, a
Highly N-Methylated Cyclic Peptide. Org. Lett., 2012, 14, 612.
[0023] [NPL 10] W.-J. Fang, et al., Deletion of Ac-NMePhe.sup.1
From [NMePhe.sup.1]arodyn Under Acidic Conditions, Part 1: Effects
of Cleavage Conditions and N-Terminal Functionality. Peptide
Science Vol. 96, 97 [0024] [NPL 11] L. A. Carpino, et al., Stepwise
Automated Solid Phase Synthesis of Naturally Occurring Peptaibols
Using FMOC Amino Acid Fluorides. J. Org. Chem., 1995, 60, 405.
[0025] [NPL 12] H. Rodriguez, et al., A convenient
microwave-enhanced solid-phase synthesis of short chain
N-methyl-rich peptides. J. Pept. Sci., 2010, 16, 136. [0026] [NPL
13] R. Roodbeen, et al., Microwave Heating in the Solid-Phase
Synthesis of N-Methylated Peptides: When Is Room Temperature
Better? Eur. J. Org. Chem., 2012, 7106.
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0027] The present inventors focused on cyclic peptides comprising
N-alkylated amino acids, which may become drug-like peptides, and
examined methods of synthesizing peptide compounds having such
features by parallel synthesis. As a result, the present inventors
have discovered that when conventional synthesis methods using TFA
is applied for cyclic peptides comprising N-alkylated amino acids
which may become drug-like peptides, the above-mentioned problems
which have been discovered for the compounds described in known
documents appeared prominently and can cause the failure in
isolating the cyclic peptides. Specifically, the present inventors
have found that obtaining the desired peptide is difficult in the
case of peptides comprising N-alkylated amino acids, since the side
reaction in which the peptide chain is cleaved becomes the major
reaction during the reaction performed under acidic conditions
using TFA as steps for cleaving off from the solid phase or for
deprotecting the protecting groups of the side chain functional
groups. The present inventors have further discovered that when
amino acids having a .beta.-hydroxy group are included in a
peptide, N- to O-acyl shift reaction also proceeds during the
reaction under the acidic conditions using TFA, and obtaining the
desired peptide was difficult. These problems were found in the
compounds of the known documents mentioned above, but the present
inventors newly discovered that these problems are similarly
observed in many other peptides. In addition to these problems, the
present inventors have also discovered a problem that when peptides
comprising an amino acid that has a hydroxy group in its skeleton
(not necessarily at the .beta. position) are subjected to reaction
under acidic conditions using TFA, its hydroxy group was esterified
with TFA.
[0028] Furthermore, the present inventors have discovered that,
when considering industrialization of peptide synthesis comprising
N-alkylated amino acids which may become drug-like peptides, the
conventional TFA-utilizing deprotection method gives considerable
difficulty in realizing industrialization not only from the
viewpoint of deprotection reaction or elongation reaction itself,
but also the subsequent work-up processes and large-scale
synthesis. For example, when the solvent of a TFA/DCM solution is
removed by condensation, the TFA concentration increases as the
condensation progresses, and problems such as hydrolysis and N- to
O-acyl shift arise simultaneously with the condensation. This could
lead to the failure in obtaining the desired compound, or cause
remarkable decrease in yield. The condensation step needs to be
performed at low temperature. Furthermore, even with low
concentration of TFA, a large excess amount of TFA compared to that
of the target molecule is included; therefore, a large excess
amount of base to be added is necessary in order to stop reactions
by neutralizing TFA, causing a large excess of salt to remain
together with the desired peptide, and this could lead to trouble
to the purification step. Furthermore, although TFA itself is a
solvent that effectively dissolves peptides, with only a lowered
concentration of TFA solution, solubility of the peptide will lead
to become low. Regarding solubility, a solvent that has high
solubility to a group of peptides needs to be selected, not only
when considering industrialization, but also in parallel synthesis
which handles many different peptide compounds at once.
[0029] In addition, the present inventors focused on improvement of
reactivity by decreasing the steric size of the protecting group of
an Fmoc-amino acid which has a protecting group-bearing functional
group on its side chain, which had so far not been undertaken with
great effort. For example, since threonine (Thr) has a hydroxyl
group, to allow the subsequent acylation reaction to occur
selectively at the amino group, a protecting group for the hydroxyl
group is necessary. However, since Thr has a branched secondary
alcohol at the p-position as its side chain functional group,
condensation efficiency of the protected Thr is relatively low due
to its bulkiness. Protecting groups generally used for Thr in
peptide synthesis include an acetyl (Ac) group, tBu group, Trt
group, benzyl (Bn) group, and t-butyldimethylsilyl (TBS) group
(Albert Isidro-Llobet, et al., Amino Acid-Protecting Groups. Chem.
Rev., 2009, 109, 2455.; Watanabe Chemical Industries, Ltd. Reagent
Catalog, Amino acids & chiral building blocks to new medicine
2012-14). However, regarding Trt group and TBS group, their
bulkiness leads to decreased condensation efficiency. Furthermore,
even in the case of tBu group which is deprotectable with acids,
condition of high TFA concentration is necessary for the
deprotection; therefore, the already mentioned problems during
deprotection become obvious. Other protecting groups are not
recognized as protecting groups that can be easily removed using
acids. To sum, it is necessary to discover protecting groups that
are sterically small enough to not reduce the condensation
efficiency and can also easily be deprotected by acids that can
avoid the above-mentioned problems of acid hydrolysis and N- to
O-acyl shift. The same broadly applies to other cases for N-methyl
serine (MeSer), which does not have a branched site at the
n-position but has higher bulkiness due to N-substitution, and for
amino acids having hydroxy groups as their functional groups.
[0030] More specifically, an objective of the present invention is
to discover novel reaction processes that can reduce the problems
of side reactions such as peptide acid hydrolysis and N- to O-acyl
shift, and TFA, esterification of hydroxy groups in the
deprotection step using TFA, which are found to become conspicuous
during parallel syntheses of peptides comprising N-substituted
amino acids, and that can also secure peptide solubility.
Furthermore, an objective of the present invention is to provide
methods of obtaining peptides comprising N-substituted amino acids
with high purity and high synthetic yield by using appropriate
protecting groups for the side-chain functional groups (appropriate
protecting groups from the viewpoint of reducing the bulkiness of
the protecting groups to improve the low reactivity during
elongation, and being deprotectable under deprotection conditions
of the present invention).
[0031] Specifically, objectives are: [0032] (1) to find reaction
conditions necessary for suppressing hydrolysis during acid
addition (during cleavage reaction from a solid phase and during
side chain deprotection reaction), particularly hydrolysis derived
from N-substituted amino groups; [0033] (2) to find reaction
conditions that enable practical work-up upon acid addition; [0034]
(3) to find reaction conditions including solvents, with
consideration on the characteristic solubility of unnatural peptide
compounds; and [0035] (4) to suppress side-reactions after
deprotection (N- to O-acyl shift and side-reactions between
hydroxyl groups and the reaction reagents, for example, TFA
acylation reaction when using TFA as the reagent) when the
unnatural peptide compounds comprise functional groups such as
hydroxyl groups, in performing parallel syntheses of N-substituted
amino acid-comprising peptide compounds having various
sequences.
[0036] In addition, an objective is to discover protecting groups
that satisfy the four conditions mentioned above for various
functional groups in amino acid side chains.
[0037] Furthermore, considering industrial production of peptide
compounds comprising N-substituted amino acids, an objective of the
present invention is to find production methods applicable to
optimization of specific sequences.
Means for Solving the Problems
[0038] In order to realize efficient synthesis of cyclic peptides
comprising N-substituted amino acids, the present inventors
discovered novel methods that can solve many problems, for example
by achieving suppression of hydrolysis and N- to O-acyl shift
progression, establishment of practical work-up methods,
suppression of TFA ester formation when hydroxyl group is present,
and selection of solvents that can secure peptide solubility, which
could not have been sufficiently solved by generally performed
improvement methods, such as the methods of decreasing the TFA
concentration or the methods of lowering the reaction temperature,
in addition to problems observed when using conventional peptide
synthesis methods that use TFA to synthesize compounds described in
known literatures. In the novel methods, TFA used in conventional
peptide synthesis is not used at all, and the present inventors
succeeded in obtaining target molecules with high selectivity.
[0039] In one embodiment of the present invention, TFA is not used
in the step of cleavage from a solid phase, and a weaker acid, for
example .2,2,2-trifluoroethanol (TFE) or hexafluoro-2-propanol
(HFIP) is used. Additionally, in another embodiment of the present
invention, protecting groups for side chain functional groups which
are not deprotected in the cleavage step are used. In the cleavage
step using an acid weaker than TFA, such as TFE or HFIP, the rates
of side reactions such as amide-bond hydrolysis are sufficiently
low, unlike the case using TFA, even during concentration step
after reaction. In particular, when using an acid weaker than TFA,
such as TFE or HFIP, the rates of side reactions are low even for
peptides comprising highly N-substituted amino acids and cyclic
peptides which are susceptible to side reactions. Therefore, the
desired compounds can be obtained as the major products. in another
embodiment of the present invention, reagents satisfying the
following conditions are used in the cleavage step: (1) the
reaction of cleavage from the solid phase proceeds smoothly while
peptide side reactions (such as hydrolysis) are suppressed; (2)
rate of side reactions is sufficiently slow even when work-up such
as concentration is performed; (3) high solubility is secured also
for highly lipid-soluble unnatural peptides; and (4) cleavage is
possible while the protecting groups of the side chain functional
groups are retained. By using a reagent that satisfies such
conditions, synthesis of peptides comprising many N-substituted
amino acids, particularly synthesis of drug-like peptides
comprising many N-alkyl groups becomes possible. Reagents
satisfying such conditions can be used not only during parallel
syntheses but also when industrially synthesizing specific
peptides.
[0040] An embodiment of the present invention provides peptide
synthesis methods which can suppress hydrolysis and N- to O-acyl
shift, and can deprotect side chain protecting groups so that the
major reaction, the desired deprotection reaction, is promoted. For
the progression of hydrolysis and N- to O-acyl shift, acid strength
(proton concentration) alone may be important. And, the inventors
have found that by using a weak acid having weakened acidity
instead of a strong acid such as TFA, progression of hydrolysis and
N- to O-acyl shift can be suppressed. Furthermore, for the
progression of desired deprotection, a step of dissociation of the
protecting groups as cationic species (carbocation or oxonium
cation) from the protected functional groups may be important, in
addition to acid strength (proton concentration). Therefore, as a
solvent that promotes the step of dissociation of protecting groups
as cationic species, the inventors have found that the use of
solvents having ionization ability can promote deprotection by the
above-mentioned weak acid.
[0041] In addition, to establish highly efficient methods for
synthesizing the drug-like peptides described in PTL 1, the
inventors discovered protecting groups for the side-chain
functional groups of amino acids having side chains with small
ionization degree under neutral conditions, which protecting groups
are not deprotected under the weakly acidic conditions used when
cleaving the peptides off from resins but can be deprotected under
the above-mentioned weak acid conditions, and which functional
groups are, for example, hydroxyl groups which are side chain
functional groups of amino acids such as Ser and Thr; alkylalcohol
groups having hydroxy groups in the side chains; phenol groups
which are side chain functional groups of amino acids such as Tyr;
imidazole groups which are side chain functional groups of amino
acids such as His; side chain carboxylic acids which are side chain
functional groups of amino acids such as Asp and Glu; and main
chain carboxylic acid of peptides or amino acids.
[0042] Furthermore, the inventors found protecting groups that can
be deprotected under the above-mentioned weak acid conditions and
can improve the low reactivity during elongation reaction, for
cases where amino acids such as .beta.-hydroxy-.alpha.-amino acids
(for example, Thr, Ser, and derivatives thereof) for which low
reactivity during elongation reaction is concerned have protecting
groups.
[0043] More specifically, the present invention is: [0044] [1] a
method of producing a peptide comprising at least one N-substituted
amino acid or N-substituted amino acid analog, wherein the method
comprises the steps of [0045] 1) preparing an amino acid
(Fmoc-protected amino acid) comprising at least one each of
following functional groups i) and ii), an amino acid analog
(Fmoc-protected amino acid analog) comprising at least one each of
following i) and ii), or a peptide (Fmoc-protected peptide)
comprising either one or both of the Fmoc-protected amino acid and
the Fmoc-protected amino acid analog: [0046] i) a main chain amino
group protected by at least one protecting group having an Fmoc
skeleton; and [0047] ii) at least one free carboxylic acid group or
active esterified carboxylic acid group; [0048] 2) making the
Fmoc-protected amino acid, the Fmoc-protected amino acid analog, or
the Fmoc-protected peptide prepared in step 1) to be supported onto
a solid phase; [0049] 3) deprotecting the protecting group having
the Fmoc skeleton of the Fmoc-protected amino acid, the
Fmoc-protected amino acid analog, or the Fmoc-protected peptide,
which is supported onto the solid phase, by using a base to expose
its amino group; [0050] 4) forming an amide bond by adding a new
Fmoc-protected amino acid, a new Fmoc-protected amino acid analog,
or a new Fmoc-protected peptide; and [0051] 5) cleaving the peptide
obtained in step 4) off from the solid phase under a condition of
weaker acidity than TFA, [0052] [2] the production method of [1],
wherein at least one side chain of the amino acid or amino acid
analog constituting the peptide obtained in step 4) is protected by
a protecting group that is not deprotected under a basic condition
but is deprotected by a first acid, and wherein the method further
comprises before or after step 5). a step of deprotecting the
protecting group using the first acid; and [0053] wherein in step
5), the peptide is cleaved off using a second acid, and wherein the
first acid and the second acid are both weaker acids than TFA and
the acidity of the first acid is higher than the acidity of the
second acid; [0054] [3] a method of producing a peptide comprising
at least one N-substituted amino acid or N-substituted amino acid
analog, wherein the method comprises the steps of: [0055] 1)
preparing an amino acid (Fmoc-protected amino acid) comprising at
least one each of following functional groups i) and ii), an amino
acid analog (Fmoc-protected amino acid analog) comprising at least
one each of following functional groups i) and ii), or a peptide
(Fmoc-protected peptide) comprising either one or both of the
Fmoc-protected amino acid and the Fmoc-protected amino acid analog:
[0056] i) a main chain amino group protected by at least one
protecting group having an Fmoc skeleton; and [0057] ii) at least
one free carboxylic acid group or active esterified carboxylic acid
group; [0058] 2) deprotecting the protecting group having the
Fmoc-skeleton of the Fmoc-protected amino acid, the Fmoc-protected
amino acid analog, or the Fmoc-protected peptide, by using a base
to expose its amino group; [0059] 3) forming an amide bond by
adding a new Fmoc-protected amino acid, a new Fmoc-protected amino
acid analog, or a new Fmoc-protected peptide, wherein at least one
side chain of the amino acid or amino acid analog constituting a
peptide obtained in this step has a protecting group that is not
deprotected under a basic condition and is deprotected under a
condition having weaker acidity than TFA; and [0060] 4)
deprotecting the protecting group of the side chain under the
condition having weaker acidity than TFA; [0061] [4] the production
method of [3], wherein peptide production is carried out by a solid
phase method; [0062] [5] the production method of [4], which
further comprises before or after step 4), a step of cleaving the
peptide obtained in step 3) off from the solid phase under a
condition further weaker than the weakly-acidic condition used in
step 4); [0063] [6] the production method of [3], wherein peptide
production is carried out by a liquid phase method; [0064] [7] the
production method of any one of [1] to [6], wherein step 4) of [1]
or step 3) of [3] further comprises the steps of [0065]
deprotecting the protecting group having the Fmoc skeleton on the
newly added Fmoc-protected amino acid, the newly added
Fmoc-protected amino acid analog, or the newly added Fmoc-protected
peptide, by using a base to expose its amino group; and [0066]
forming an amide bond by further adding a new Fmoc-protected amino
acid, a new Fmoc-protected amino acid analog, or a new
Fmoc-protected peptide, and wherein these steps are repeated once
or multiple times; [0067] [8] the production method of any one of
[1] to [7], wherein the produced peptide comprises on its
C-terminal side an amino acid residue or an amino acid analogue
residue comprising one reactive site, and comprises on its
N-terminal side an amino acid residue, an amino acid analogue
residue, or a carboxylic acid analog comprising the other reactive
site; [0068] [9] the production method of [8], which further
comprises the step of bonding said reactive site and said other
reactive site to cyclize the peptide; [0069] [10] the production
method of [9], wherein the amino acid residue, the amino acid
analogue residue, or the carboxylic acid analog having said other
reactive site is at the N terminus and the bonding is an amide
bonding; [0070] [11] the production method of [9], wherein the
amino acid residue, the amino acid analogue residue, or the
carboxylic acid analog having said other reactive site is at the N
terminus and the bonding is a carbon-carbon bonding; [0071] [12]
the production method of any one of [1] to [11], wherein the step
performed under a condition having weaker acidity than TFA is
performed using a weakly acidic solution comprising a weak acid
having an aqueous pKa value of 0 to 9 in a solvent having an
aqueous pKa value of 5 to 14 and whose ionization ability value
YOTs is positive; [0072] [13] the production method of [12],
wherein the solvent is fluoroalcohol; [0073] [14] the production
method of [13], wherein the fluoroalcohol is TFE or HFIP; [0074]
[15] the production method of any one of [2] to [14], wherein the
side chain protecting group is a protecting group which is
deprotected in the range of pH 1 to pH 7, or a protecting group
which is deprotected in 10% or lower concentration of TFA; and
[0075] [16] the production method of any one of [2] to [15],
wherein the side chain protecting group is selected from following
a) to d): [0076] a) when the side chain protecting group is a
protecting group for the side chain hydroxyl group of Ser, Thr, Hyp
(hydroxyproline), and derivatives thereof, any one protecting group
selected from a MOM skeleton, a Bn skeleton, a Dpm skeleton, a Trt
skeleton, a silyl skeleton, and a Boc skeleton represented by the
general formulae below; [0077] b) when the side chain protecting
group is a protecting group for the side chain hydroxyl group of
Tyr and derivatives thereof, any one protecting group selected from
a MOM skeleton, a Bn skeleton, a Dpm skeleton, a Trt skeleton, a
silyl skeleton, a Boc skeleton, and a tBu skeleton represented by
the general formulae below; [0078] c) when the side chain
protecting group is a protecting group for the side chain imidazole
ring of His and derivatives thereof, any one protecting group
selected from a MOM skeleton, a Bn skeleton, and a Trt skeleton
represented by the general formulae below; and [0079] d) when the
side chain protecting group is a protecting group for the side
chain carboxylic acid group of Asp, Glu, and derivatives thereof,
any one protecting group selected from a MOM skeleton, a Bn
skeleton, a Dpm skeleton, a Trt skeleton, a tBu skeleton, a
phenyl-EDOTn skeleton, which are represented by the following
general formulae, and an orthoester skeleton in which a carbon atom
of the carboxylic acid group to be protected is substituted with
three alkoxy groups: [0080] <a protecting group having a MOM
skeleton>
[0080] ##STR00001## [0081] (wherein [0082] R1 is H, R2 is H, and X
is methyl, benzyl, 4-methoxybenzyl, 2,4-dimethoxybenzyl,
3,4-dimethoxybenzyl, or 2-trimethylsilylethyl; [0083] R1 is methyl,
R2 is H, and X is ethyl; [0084] R1, R2, and R3 are all methyl; or
[0085] R1 and X together form --CH.sub.2--CH.sub.2--CH.sub.2-- or
--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--, and R2 is H, [0086]
wherein when any one of R1, R2, and X is methyl or ethyl, these
groups may further be substituted with alkyl, benzyl, or aryl);
[0087] <a protecting group having a Bn skeleton>
[0087] ##STR00002## [0088] (wherein [0089] R1 to R5 are each
independently H, alkyl, aryl, or halogen, and R6 and R7 are alkyl;
[0090] R1, R2, R4, and R5 are each independently H, alkyl, aryl, or
halogen, R3 is methoxy, and R6 and R7 are H; [0091] R1 and R3 are
methoxy, R2, R4, and R5 are each independently H, alkyl, aryl, or
halogen, and R6 and R7 are H; or [0092] R1, R4, and R5 are each
independently H, alkyl, aryl, or halogen, and R2 and R3 together
form --O--CH2-O--); [0093] <a protecting group having a Dpm
skeleton>
[0093] ##STR00003## [0094] (wherein [0095] R1 to R10 are each
independently H, alkyl, aryl, alkoxy, or halogen; or [0096] R1 to
R4 and R7 to R10 are each independently H, alkyl, aryl, alkoxy, or
halogen, and R5 and R6 together form --O-- or --CH2-CH2-); [0097]
<a protecting group having a Trt skeleton>
[0097] ##STR00004## [0098] (wherein [0099] R1 to R15 are each
independently H, alkyl, aryl, alkoxy, or halogen; [0100] R1, R2,
and R4 to R15 are each independently H, alkyl, aryl, alkoxy, or
halogen, and R3 is methyl or methoxy; [0101] R1 is Cl, and R2 to
R15 are each independently H, alkyl, aryl, alkoxy or halogen; or
[0102] R1 to R4 and R7 to R15 are each independently H, alkyl,
aryl, alkoxy, or halogen, and R5 and R6 together form --O--);
[0103] <a protecting group having a silyl skeleton>
[0103] ##STR00005## [0104] (wherein R1 to R3 are each independently
alkyl or aryl); [0105] <a protecting group having a Boc
skeleton>
[0105] ##STR00006## [0106] (wherein R1 to R9 are each independently
H, alkyl, or aryl); [0107] <a protecting group having a tBu
skeleton>
[0107] ##STR00007## [0108] (wherein [0109] R1 to R9 are each
independently H, alkyl, or aryl); and [0110] <a protecting group
having a phenyl-EDOTn skeleton>
[0110] ##STR00008## [0111] (wherein R1 to R3 are each independently
H or methoxy).
Effects of the Invention
[0112] Peptides comprising N-substituted amino acids can be
obtained with high synthesis efficiency and high purity by the
present invention.
[0113] For example, in the case of peptide sequences comprising
amino acids having protecting groups on their side chains, [0114]
(1) the combination of an acid weaker than TFA and a solvent
showing ionizing ability discovered by the present invention can
allow deprotection to be carried out with minimized acid hydrolysis
of the peptide chains, and with minimized N- to O-acyl shift, TFA
esterification, and such which may occur for sequences comprising
p-hydroxy-a-amino acids (for example, Ser, Thr, and derivatives
thereof), and [0115] (2) when elongating the amino acids by amide
bond-forming reactions, the reaction rate and reaction efficiency
can be improved in comparison to when amino acids have protecting
groups used for general peptide synthesis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0116] FIG. 1 shows the basic synthetic route for a cyclic peptide
comprising an N-methyl amino acid in its sequence.
[0117] FIG. 2 shows the result showing detection of the desired
peptide (Compound 131), hydrolysate of the target molecule
(TM+H2O), and a solvolysis product of the target molecule by HFIP
(TM+HFIP) under the deprotection condition using 0.1 M
tetramethylammonium hydrogensulfate/HFIP solution (2% TIPS)
analyzed by LCMS.
[0118] FIG. 3 shows the result showing detection of the desired
peptide (Compound 131), hydrolysate of the target molecule
(TM+H2O), and a solvolysis product of the target molecule by HFIP
(TM+HFIP) under the deprotection condition using 0.05 M
tetramethylammonium hydrogensulfate/HFIP solution (2% TIPS)
analyzed by LCMS.
[0119] FIG. 4 shows the result showing detection of the desired
peptide (Compound 133), and a N- to O-acyl shifted product of
desired product under the deprotection condition using 0.05 M
tetramethylammonium hydrogensulfate/HFIP solution (2% TIPS)
analyzed by LCMS.
[0120] FIG. 5 shows the result showing detection of the desired
peptide (Compound 131), hydrolysate of the target molecule
(TM+H2O), and a solvolysis product of the target molecule by HFIP
(TM+HFIP) under the deprotection condition using 0.05 M oxalic
acid/HFIP solution (2% TIPS) analyzed by LCMS.
[0121] FIG. 6 shows the result showing detection of the desired
peptide (Compound 131), hydrolysate of the target molecule
(TM+H2O), and a solvolysis product of the target molecule by HFIP
(TM+HFIP) under the deprotection condition using 0.05 M maleic
acid/HFIP solution (2% TIPS) analyzed by LCMS.
[0122] FIG. 7 shows the result showing detection of the desired
peptide (Compound 133), and a N- to O-acyl shifted product of the
target molecule under the deprotection condition using 0.05 M
oxalic acid/HFIP solution (2% TIPS) analyzed by LCMS.
[0123] FIG. 8 shows the result showing detection of the desired
peptide (Compound 133), and a N- to O-acyl shifted product of the
target molecule under the deprotection condition using 0.05 M
maleic acid/HFIP solution (2% TIPS) analyzed by LCMS.
[0124] FIG. 9 shows the result showing detection of the desired
peptide (Compound 137) and a solvolysis product of the target
molecule by HFIP (product in which any one of the amide bonds has
undergone solvolysis by HFIP) under the deprotection condition
using 0.05 M tetramethylammonium hydrogensulfate/HFIP (2% TIPS)
analyzed by LCMS.
[0125] FIG. 10 shows the results showing detection of the desired
peptide (Compound 137) and solvolysis product of the target
molecule by TFE (product in which any one of the amide bonds has
undergone solvolysis by TFE) under the deprotection condition using
0.05 M tetramethylammonium hydrogensulfate/TFE (2% TIPS) analyzed
by LCMS.
[0126] FIG. 11 shows the results showing detection of the desired
peptide (Compound 135) analyzed by LCMS, when 0.1 M
tetramethylammonium hydrogen sulfate/HFIP solution (2% TIPS) was
used as the deprotection condition, and a base (DIPEA) was added to
this solution to stop the reaction.
[0127] FIG. 12 shows the results showing detection of the desired
peptide (Compound 133) analyzed by LCMS, when 0.1 M
tetramethylammonium hydrogensulfate/HFIP solution (2% TIPS) was
used as the deprotection condition, and a base (DIPEA) was added to
this solution to stop the reaction.
[0128] FIG. 13 shows the results showing detection of the desired
peptide (Compound 112) and a product in which Thr has been removed
from the desired peptide (Compound 113) analyzed by LCMS, when
Fmoc-Thr(Trt)-OH was added.
[0129] FIG. 14 shows the results showing detection of the desired
peptide (Compound 114) analyzed by LCMS when Fmoc-Thr(THP)-OH was
added. The product (Compound 113) in which Thr was missing from the
desired peptide (Compound 114) was not detected.
[0130] FIG. 15 shows the results showing detection of the desired
peptide (Compound 115) and the product (Compound 116) in which
MeSer was missing from the desired peptide analyzed by LCMS, when
the synthesis was carried out using Fmoc-MeSer(DMT)-OH0.75
DIPEA.
[0131] FIG. 16 shows the results showing detection of the desired
peptide (Compound 115) and the product (Compound 116) in which
MeSer was missing from the desired peptide analyzed by LCMS, when
the synthesis was carried out using Fmoc-MeSer(THP)-OH (Compound
6).
[0132] FIG. 17 shows the result showing detection of the desired
peptide (Compound 131) and hydrolysate of the target molecule
(TM+H2O) under the deprotection condition using 5% TFA/DCE (5%
TIPS) analyzed by LCMS.
[0133] FIG. 18 shows the result showing detection of the desired
peptide (Compound 133), a N- to O-acyl shifted product of the
target molecule, a compound in which one hydroxyl group of the
target molecule has been TFA esterified, and a compound in which
two hydroxyl groups of the target molecule have been TFA esterified
under the deprotection condition using 5% TFA/DCE (5% TIPS)
analyzed by LCMS.
[0134] FIG. 19 shows the result showing detection of the desired
peptide (Compound 133), a N- to O-acyl shifted product of the
target molecule, a compound in which one hydroxyl group of the
target molecule has been TFA esterified, and a compound in which
two hydroxyl groups of the target molecule have been TFA esterified
under the deprotection condition using 5% TFA/DCE (5% TIPS)
(0.degree. C.) analyzed by LCMS.
[0135] FIG. 20 shows the result showing detection of the desired
peptide (Compound 133), a N- to O-acyl shifted product of the
target molecule, a compound in which one hydroxyl group of the
target molecule has been TFA esterified, and a compound in which
two hydroxyl groups of the target molecule have been TFA esterified
wider the deprotection condition using 5% TFA/DCE (5% TIPS)
(25.degree. C.) analyzed by LCMS.
[0136] FIG. 21 shows the synthesis method including elongation
reaction in the liquid phase.
MODE FOR CARRYING OUT THE INVENTION
[0137] In a certain embodiment, the present invention relates to
methods of producing a peptide comprising at least one
N-substituted amino acid or N-substituted amino acid analog,
wherein the method comprises the steps of: [0138] 1) preparing an
amino acid (Fmoc-protected amino acid) having at least one each of
following functional groups i) and ii), an amino acid analog
(Fmoc-protected amino acid analog) having at least one each of
following i) and ii), or a peptide (Fmoc-protected peptide)
comprising either one or both of the Fmoc-protected amino acid and
the Fmoc-protected amino acid analog: [0139] i) a main chain amino
group protected by at least one protecting group having an Fmoc
skeleton; and [0140] ii) at least one free carboxylic acid group or
active esterified carboxylic acid group; [0141] 2) making the
Fmoc-protected amino acid, the Fmoc-protected amino acid analog, or
the Fmoc-protected peptide prepared in step 1) to be supported onto
a solid phase; [0142] 3) deprotecting the protecting group having
an Fmoc skeleton of the Fmoc-protected amino acid, the
Fmoc-protected amino acid analog, or the Fmoc-protected peptide,
which is supported onto the solid phase, by using a base to expose
its amino group; [0143] 4) forming an amide bond by adding a new
Fmoc-protected amino acid, a new Fmoc-protected amino acid analog,
or a new Fmoc-protected peptide; and [0144] 5) cleaving the peptide
obtained in step 4) off from the solid phase under a condition of
weaker acidity than TFA.
[0145] In another embodiment, the present invention relates to a
method of producing a peptide comprising at least one N-substituted
amino acid or N-substituted amino acid analog, wherein the method
comprises the steps of: [0146] 1) preparing an amino acid
(Fmoc-protected amino acid) having at least one each of following
functional groups i) and ii), an amino acid analog (Fmoc-protected
amino acid analog) having at least one each of following functional
groups i) and ii), or a peptide (Fmoc-protected peptide) comprising
either one or both of the Fmoc-protected amino acid and the
Fmoc-protected amino acid analog: [0147] i) a main chain amino
group protected by at least one protecting group having an Fmoc
skeleton; and [0148] ii) at least one free carboxylic acid group or
active esterified carboxylic acid group; [0149] 2) deprotecting the
protecting group having an Fmoc skeleton of the Fmoc-protected
amino acid, the Fmoc-protected amino acid analog, or the
Fmoc-protected peptide, by using a base to expose its amino group;
[0150] 3) forming an amide bond by adding a new Fmoc-protected
amino acid, a new Fmoc-protected amino acid analog, or a new
Fmoc-protected peptide, wherein at least one side chain of the
amino acid or amino acid analog constituting a peptide obtained in
this step has a protecting group that is not deprotected under a
basic condition and is deprotected under a condition having weaker
acidity than TFA; and [0151] 4) deprotecting the protecting group
of the side chain under a condition having weaker acidity than
TFA.
[0152] The above-mentioned peptide production may be performed by a
solid phase method or a liquid phase method.
[0153] "Peptide" in the present invention is not particularly
limited as long as it is a peptide formed by amide bonding or ester
bonding of amino acids and/or amino acid analogs, and is preferably
a peptide of 5 to 30 residues, more preferably 7 to 15 residues,
and even more preferably 9 to 13 residues, Peptides synthesized in
the present invention comprise at least one or more amino acids or
amino acid analogs which have been N-substituted (also called
N-substituted amino acids), and preferably comprise two or more,
more preferably three or more, and even more preferably five or
more N-substituted amino acids, in a single peptide. These
N-substituted amino acids may be present consecutively or
non-consecutively in a peptide.
[0154] Peptides in the present invention may be linear peptides or
cyclic peptides, and are preferably cyclic peptides.
[0155] A "cyclic peptide" in the present invention can be obtained
by synthesizing a linear peptide according to methods of the
present invention, and then cyclizing it. The cyclization may be in
any form such as cyclization by a carbon-nitrogen bonding such as
an amide bonding, cyclization by a carbon-oxygen bonding such as an
ester bonding or an ether bonding, cyclization by a carbon-sulfur
bonding such as a thioether bonding, cyclization by a carbon-carbon
bonding, or cyclization by construction of a heterocycle. While not
particularly limited thereto, cyclization via a covalent bonding
such as a carbon-carbon bonding or an amide bonding is preferred,
and cyclization via an amide bonding formed by a side chain
carboxylic acid group and an N-terminal main chain amino group is
particularly preferred. The sites of the carboxylic acid group,
amino group, and such used in the cyclization may be on the main
chain or on the side chain, and are not particularly limited as
long as they are at sites where cyclization is possible.
[0156] An "N-substituted amino acid" in the present invention means
an amino acid or an amino acid analog in which the main chain amino
group of the later described "amino acid" or "amino acid analog" is
N-substituted, and an amino acid or amino acid analog that is
N-alkylated, such as N-methylated, is preferred. Specific examples
of an N-substituted amino acid include amino acids or amino acid
analogs in which the main chain amino group is an NHR group,
wherein R is an optionally substituted alkyl group, optionally
substituted alkenyl group, optionally substituted alkynyl group,
optionally substituted aryl group, optionally substituted
heteroaryl group, optionally substituted aralkyl group, or
optionally substituted cycloalkyl group, or alternatively those in
which a carbon atom bonded to the N atom forms a ring with a carbon
atom from the a position such as proline. The substituent of each
of the optionally substituted groups is not particularly limited,
and examples include a halogen group, an ether group, and a
hydroxyl group.
[0157] Specifically, for such N-substituted amino acids, an alkyl
group, an aralkyl group, a cycloalkyl group, or such are preferably
used.
[0158] An "amino acid" in the present invention is .alpha.-,
.beta.-, and .gamma.-amino acids, and is not limited to natural
amino acids and may be unnatural amino acids. (In the present
invention, "natural amino acids" refer to the 20 types of amino
acids included in proteins. Specifically, they refer to Gly, Ala,
Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gin, Asn,
Cys, Met, Lys, Arg, and Pro.) In the case of .alpha.-amino acids,
they may be L-amino acids or D-amino acids, or may be
.alpha.,.alpha.-dialkylamino acids. Selection of amino acid side
chains are not particularly limited, but examples include a
hydrogen atom, alkyl groups, alkenyl groups, alkynyl groups, aryl
groups, heteroaryl groups, aralkyl groups, and cycloalkyl groups.
The amino acid side chains may be respectively attached with
substituent groups, and substituent groups are freely selected from
among any functional groups including, for example, an N atom, an O
atom, an S atom, a B atom, a Si atom, or a P atom. The number of
substituent groups is not particularly limited and the amino acid
side chains may have one or two or more substituent groups.
[0159] The term "amino acid analog" in the present invention
preferably means .alpha.-hydroxycarboxylic acids. Like amino acids,
the side chains of .alpha.-hydroxycarboxylic acids are not
particularly limited, and examples include a hydrogen atom, alkyl
groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl
groups, aralkyl groups, and cycloalkyl groups. The steric
structures of a-hydroxycarboxylic acids may be those that
correspond to the L- or D-form of amino acids. The side chains are
not particularly limited, and are freely selected from among
arbitrary functional groups carrying, for example, an N atom, an O
atom, an S atom, a B atom, a Si atom, or a P atom. They may have
one or two or more substituent groups, and the number of
substituent groups is not particularly limited. For example, they
may have an S atom, and may also have functional groups such as
amino groups or halogen groups. Similarly to the case with a-amino
acids, arbitrary steric configurations are accepted in the case of
.beta.- and .gamma.-amino acids as well, and selection of their
side chains is also not particularly limited.
[0160] The "amino acids" or "amino acid analogs" constituting the
peptides synthesized in the present invention includes all their
respective corresponding isotopes. The isotope in the "amino acids"
or "amino acid analogs" refers to one in which at least one atom is
replaced with an atom f the same atomic number (number of protons
is the same) and of different mass number (sum of the number of
protons and neutrons is different). Examples of the isotope
contained in the "amino acids" or "amino acid analogs" constituting
the peptide compounds of the present invention include a hydrogen
atom, a carbon atom, a nitrogen atom, an oxygen atom, a phosphorus
atom, a sulfur atom, a fluorine atom and a chlorine atom, and
specific examples include 2H, 3H, 13C, 14C, 15N, 17O, 18O, 31P,
32P, 35S, 18F, and 36Cl.
[0161] The amino acids or the amino acid analogs may have one or
two or more substituent groups. Examples of such substituent groups
include those derived from an O atom, an N atom, an S atom, a B
atom, a P atom, a Si atom, and a halogen atom.
[0162] Examples of halogen-derived substituents include fluoro
(--F), chloro (--Cl), bromo (--Br), and iodo (--I).
[0163] Examples of O atom-derived substituents include hydroxyl
(--OH), oxy (--OR), carbonyl (--C.dbd.O--R), carboxyl (--CO2H),
oxycarbonyl (--C.dbd.O--OR), carbonyloxy (--O--C.dbd.O--R),
thiocarbonyl (--C.dbd.O--SR), carbonylthio group (--S--C.dbd.O--R),
aminocarbonyl (--C.dbd.O--NHR), carbonylamino (--NH--C.dbd.O--R),
oxycarbonylamino (--NH--C.dbd.O--OR), sulfonylamino (--NH--SO2-R),
aminosulfonyl (--SO2-NHR), sulfamoylamino (--NH--SO2-NHR),
thiocarboxyl (--C(.dbd.O)--SH), carboxylcarbonyl
(--C(.dbd.O)--CO2H).
[0164] Examples of oxy (--OR) include alkoxy, cycloalkoxy,
alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, and aralkyloxy.
[0165] Examples of carbonyl (--C.dbd.O--R) include formyl
(--C.dbd.O--H), alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl,
alkynylcarbonyl, arylcarbonyl, heteroarcarbonyl, and
aralkylcarbonyl.
[0166] Examples of oxycarbonyl (--C.dbd.O--OR) include
alkyloxycarbonyl, cycloalkyloxycarbonyl, alkenyloxycarbonyl,
alkynyloxycarb aryloxycarbonyl, heteroaryloxycarbonyl, and
aralkyloxycarbonyl.
[0167] (--C.dbd.O--OR)
[0168] Examples of carbonyloxy (--O--C.dbd.O--R) include
alkylcarbonyloxy, cycloalkylcarbonyloxy, alkenylcarbonyloxy,
akynylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, and
aralkylcarbonyloxy.
[0169] Examples of thiocarbonyl(--C.dbd.O--SR) include
alkylthiocarbonyl, cycloalkylthiocarbonyl, alkenylthiocarbonyl,
alkynylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, and
aralkylthiocarbonyl.
[0170] Examples of carbonylthio (--S--C.dbd.O--R) include,
alkylcarbonylthio, cycloalkylcarbonylthio, alkenylcarbonylthio,
alkynylcarbonylthio, arylcarbonylthio, heteroarylcarbonylthio, and
aralkylcarbonylthio.
[0171] Examples of aminocarbonyl (--C.dbd.O--NHR) include
alkylaminocarbonyl, cycloalkylaminocarbonyl, alkenylaminocarbonyl,
alkynylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl,
and aralkylaminocarbonyl. Additional examples include compounds
produced by further substitution of an alkyl, a cycloalkyl, an
alkenyl, an alkynyl, an aryl, a heteroaryl, or an aralkyl for the H
atom bonded to the N atom in --C.dbd.O--NHR.
[0172] Examples of carbonylamino (--NH--C.dbd.O--R) include
alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino,
alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino,
and aralkylcarbonylamino. Additional examples include compounds
produced by further substitution of an alkyl, a cycloalkyl, an
alkenyl, an alkynyl, an aryl, a heteroaryl, or an aralkyl for the H
atom bonded to the N atom in --NH--C.dbd.O--R.
[0173] Examples of oxycarbonylamino (--NH--C.dbd.O--OR) include
alkoxycarbonylamino, cycloalkoxycarbonylamino,
alkenyloxycarbonylamino, alkynyloxycarbonylamino,
aryloxycarbonylamino, heteroaryloxycarbonylamino, and
aralkyloxycarbonylamino. Additional examples include compounds
produced by further substitution of an alkyl, a cycloalkyl, an
alkenyl, an alkynyl, an aryl, a heteroaryl, or an aralkyl for the H
atom bonded to the N atom in --NH--C.dbd.O--OR.
[0174] Examples of sulfonylamino (--NH--SO2-R) include
alkylsulfonylamino, cycloalkylsulfonylamino, alkenylsulfonylamino,
alkynylsulfonylamino, arylsulfonyl amino, heteroarylsulfonylamino,
and aralkylsulfonylamino. Additional examples include compounds
produced by further substitution of an alkyl, a cycloalkyl, an
alkenyl, an alkynyl, an aryl, a heteroaryl or an aralkyl for the H
atom bonded to the N atom in --NH--SO2-R.
[0175] Examples of aminosulfonyl (--SO2-NHR) include
alkylaminosulfonyl, cycloalkylaminosulfonyl, alkenylaminosulfonyl,
alkynylaminosulfonyl, arylaminosulfonyl heteroarylaminosulfonyl,
and aralkylaminosulfonyl. Additional examples include compounds
produced by further substitution of an alkyl, a cycloalkyl, an
alkenyl, an alkynyl, an aryl, a heteroaryl, or an aralkyl for the H
atom bonded to the N atom in --SO2-NHR.
[0176] Examples of sulfamoylamino (--NH--SO2-NHR) include
alkylsulfamoylamino, cycloalkylsulfamoylamino,
alkenylsulfamoylamino, alkynylsulfamoylamino, arylsulfamoylamino,
heteroarylsulfamoylamino, and aralkylsulfamoylamino. Additionally,
the two H atoms bonded to the N atoms in --NH--SO2-NHR may be
substituted with a substituent independently selected from the
group consisting of an alkyl, a cycloalkyl, an alkenyl, an alkynyl,
an aryl, a heteroaryl, and an aralkyl; or these two substituents
may form a ring.
[0177] For S atom-derived substituents, examples include thiol
(--SH), thio (--S--R), sulfinyl (--S.dbd.O--R), sulfonyl
(--S(O)2-R), and sulfo (--SO3H).
[0178] Examples of thio (--S--R) are selected from alkylthio,
cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio,
aralkylthio, and such.
[0179] Examples of sulfinyl (--S.dbd.O--R) include alkylfulfinyl,
cycloalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, arylsulfinyl,
heteroarylsulfinyl, and aralkylsulfinyl.
[0180] Examples of sulfonyl (--S(O)2-R) include alkylsulfonyl,
cycloalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl,
heteroarylsulfonyl, and aralkylsulfonyl.
[0181] For N atom-derived substituents, examples include azide
(--N3, also called "azido group"), cyano (--CN), primary amino
(--NH2), secondary amino (--NH--R), tertiary amino (--NR(R')),
amidino (--C(.dbd.NH)--NH2), substituted amidino
(--C(.dbd.NR)--NR'R''), guanidino (--NH--C(.dbd.NH)--NH2),
substituted guanidino (--NR--C(.dbd.NR''')--NR'R''), and
aminocarbonylamino (--NR--CO--NR'R'').
[0182] Examples of secondary amino (--NH--R) include alkylamino,
cycloalkylamino, alkenylamino, alkynylamino, arylamino,
heteroarylamino, and aralkylamino.
[0183] Examples of tertiary amino (--NR(R')) include amino groups,
such as alkyl(aralkyl)amino, having any two substituents each
independently selected from alkyl, cycloalkyl, alkenyl, alkynyl,
aryl, heteroaryl, and aralkyl; and these two arbitrary substituents
may form a ring.
[0184] Examples of substituted amidino (--C(.dbd.NR)--NR'R'')
include groups in which each of the three substituents R, R', and
R'' on the N atoms is independently selected from among alkyl,
cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl; and
such examples include alkyl(aralkyl)(aryl)amidino.
[0185] Examples of substituted guanidino
(--NR--C(.dbd.NR''')--NR'R'') include groups in which each of R,
R', R'', and R''' is independently selected from alkyl, cycloalkyl,
alkenyl, alkynyl, aryl, heteroaryl, and aralkyl; or groups in which
they form a ring.
[0186] Examples of aminocarbonylamino (--NR--CO--NR'R'') include
groups in which each of R, R', and R'' is independently selected
from a hydrogen atom, alkyl, cycloalkyl, alkenyl, alkynyl, aryl,
heteroaryl, and aralkyl; or groups in which they form a ring.
[0187] Examples of B atom-derived substituents include boryl
(--BR(R')) and dioxyboryl (--B(OR)(OR')). These two substituents, R
and R', are each independently selected from alkyl, cycloalkyl,
alkenyl, alkynyl, aryl, heteroaryl, and aralkyl; or they may form a
ring.
[0188] This way, the amino acids or amino acid analogs of the
present invention may have one or two or more of the various
substituent groups including an O atom, an N atom, an S atom, a B
atom, a P atom, an Si atom, and a halogen atom, which are
ordinarily used in small molecule compounds. These substituent
groups may further be substituted with other substituent
groups.
[0189] Herein, the "amino acid" and "amino acid analog"
constituting the peptides synthesized in the present invention are
also referred to as "amino acid residue" and "amino acid analogue
residue", respectively.
[0190] In the present invention, "Fmoc-protected amino acid" and
"Fmoc-protected amino acid analog" are an amino acid and an amino
acid analog respectively having at least one each of following
functional groups i) and ii): [0191] i) a main chain amino group
protected by at least one protecting group having an Fmoc skeleton;
and [0192] ii) at least one free carboxylic acid group or active
esterified carboxylic acid group.
[0193] "Protecting group having an Fmoc skeleton" in the present
invention means an Fmoc group or a group formed by introducing
arbitrary substituent group(s) into arbitrary position(s) in the
skeleton constituting the Fmoc group. Specific examples of
protecting groups having an Fmoc skeleton include
9-fluorenylmethyloxycarbonyl (Fmoc) group, 2,7-di-tert-butyl-Fmoc
(Fmoc*) group, 2-fluoro-Fmoc (Fmoc(2F)) group, 2-monoisooctyl-Fmoc
(mio-Fmoc) group, and 2,7-diisooctyl-Fmoc (dio-Fmoc) group. In the
present invention, protecting groups that are deprotectable (that
can be deprotected) under basic conditions or by nucleophiles
showing basicity (for example, piperidine or hydrazine) may also be
used in place of protecting groups having an Fmoc skeleton.
Specific examples of such protecting groups include
2-(4-nitrophenylsulfonyl)ethoxycarbonyl (Nsc) group,
(1,1-dioxobenzo[b]thiphene-2-yl)methyloxycarbonyl (Bsmoc) group,
(1,1 -dioxonaphtho[1,2-b]thiophene-2-yl)methyloxycarbonyl
(.alpha.-Nsmoc) group,
1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidine)-3-methylbutyl (ivDde)
group, tetrachlorophthaloyl (TCP) group,
2-[phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate (Pms)
group, ethanesulfonylethoxycarbonyl (Esc) group, and
2-(4-sulfophenylsulfonyl)ethoxycarbonyl (Sps) group. Furthermore,
protecting groups that are deprotectable by means other than acids
or bases may also be used. Specific examples of such protecting
groups include benzyloxycarbonyl (Z) group which are deprotectable
by hydrogenation in the presence of a transition metal catalyst
such as palladium, allyloxycarbonyl (Alloc) group which are
deprotectable by a combination of a palladium catalyst and a
scavenger (for example, the combination of
tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) and
phenylsilane), o-nitrobenzenesulfonyl (oNBS, Ns) group which are
deprotectable by a combination of an alkylthiol or arylthiol with a
base, 2,4-dinitrobenzenesulfonyl (dNBS) group, and dithiasuccinoyl
(Dts) group, and p-nitrobenzyloxycarbonyl (pNZ) group which are
deprotectable reductively by a reducing agent such as sodium
dithionite (Na2S2O4) or by hydrogenation in the presence of a
transition metal catalyst (Reference Document: Amino
Acid-Protecting Groups, Chem. Rev. 2009, 109, 2455-2504).
[0194] In the present invention which uses the Fmoc method,
Fmoc-protected amino acids or Fmoc-protected amino acid analogs
that may preferably be used are, for example, those in which the
main chain amino group is protected by the Fmoc group, side chain
functional group(s) are protected when necessary by protecting
group(s) that are not cleaved by bases such as piperidine or DRU,
and the main chain carboxylic acid group is not protected.
Fmoc-protected amino acids or Fmoc-protected amino acid analogs,
which have an amino group protected with a protecting group that
has the Fmoc skeleton and a carboxylic acid group without a
protecting group, may also be preferably used.
[0195] In the present invention, when an Fmoc-protected amino acid,
an Fmoc-protected amino acid analog, or an Fmoc-protected peptide
has side-chain functional group(s), such functional group(s) are
preferably protected by protecting group(s). When side-chain
functional groups are protected by protecting groups, well-known
protecting groups that can be deprotected under conditions of
choice can be used. Such protecting groups are preferably
protecting groups that are not cleaved under basic conditions and
are deprotected under conditions having weaker acidity than TFA.
Protecting groups that are deprotectable under acidic conditions
include protecting groups that can be deprotected in the range of,
for example, pH 1 to pH 7, and preferably pH 2 to pH 6.
Alternatively, protecting groups that are deprotectable by 10% or
lower TFA, or protecting groups having the later-described
structures can be used. In the present invention, well-known
protecting groups can be used as the side-chain protecting groups.
For example, from among the protecting groups described in the
following documents i) and ii), those that satisfy the
above-mentioned conditions can be employed as the side-chain
protecting groups. [0196] NPL i) Greene's Protective Groups in
Organic Synthesis, Fourth Edition [0197] NPL ii) Chemical Reviews,
2009, 109(6), 2455-2504.
[0198] The methods of the present invention can be used in peptide
synthesis by parallel synthesis. In such cases, protecting groups
are not always necessary for the amino acid side chains, but when
protecting groups are necessary for the side chains, it is
preferable that the protecting groups used be deprotected quickly
under the deprotection conditions of the present invention. It is
preferable that the side chain protecting groups be 50% deprotected
in 24 hours or less, and it is particularly preferable that the
side chain protecting groups be 90% deprotected in 4 hours or less.
As protecting groups that satisfy such conditions, protecting
groups having the later-described Trt skeleton, THP skeleton, THF
skeleton, or TBS skeleton are preferred. To enable easy
deprotection by an acid and to secure high reactivity during
elongation, protecting groups having at least one hydrogen-atom
substituent on the protecting-group atom which directly bonds to
the functional group (three-dimensional bulkiness is smaller than
protecting groups having a Trt skeleton) are preferred. Among them,
protecting groups in which substituent groups other than hydrogen
form a ring are more preferred, and THP and THF are particularly
preferred.
[0199] The methods of the present invention can be used for
industrial peptide synthesis as well. In such cases too, the amino
acid side chain(s) do not always need to have protecting group(s)
as in the case of parallel synthesis, but when the side chains have
protecting groups, they preferably have the same protecting groups
as in parallel synthesis. If there is no problem with hydrolysis
and N- to O-acyl shift during deprotection for the sequence of a
peptide to be synthesized, but elongation reaction is problematic
due to the bulkiness of the protecting groups, strong acids
generally used for deprotection such as TFA may be used in
deprotection. Furthermore, when there is no problem with elongation
reaction of the peptide to be synthesized, bulky protecting groups
may also be used.
[0200] In the present invention, "a condition of weaker acidity
than TFA" preferably includes conditions that use a weakly acidic
solution containing a weak acid which has an aqueous pKa value of 0
to 9 in a solvent which has an aqueous pKa value of 5 to 14 and
whose ionization ability value YOTs is positive.
[0201] The "weak acid having an aqueous pKa value of 0 to 9" is
more preferably weak acids having an aqueous pKa of 1 to 5. Such
weak acids specifically include tetramethylammonium hydrogensulfate
(aqueous pKa=2.0), oxalic acid (aqueous pKa=1.23), and maleic acid
(aqueous pKa=1.92). The concentration of the weak acid dissolved in
a solvent may be any concentration as long as the condition of
showing weaker acidity than TFA is satisfied.
[0202] A "solvent having an aqueous pKa value of 5 to 14 and whose
ionization ability value YOTs is positive" preferably includes
fluoroalcohols. Fluoroalcohol is a generic term for an alcohol in
which a fluorine atom(s) is bonded to carbon atom(s) of the
alcohol-constituting carbon atoms excluding the carbon atom to
which a hydroxyl group is bonded. In the present invention,
alcohols in which a hydroxyl group is bonded to an aromatic ring
such as 2,3,4,5,6-pentafluorophenol are also included in the
fluoroalcohols. The preferred fluoroalcohols are
2,2,2-trifluoroethanol (TFE) and hexafluoro-2-propanol (HFIP).
[0203] In the present invention, as long as the condition of
yielding weaker acidity than TFA is satisfied, other organic
solvents (for example, dichloromethane or 1,2-dichloroethane) and
cationic scavengers (for example, triisopropylsilane), and such may
further be added to the aforementioned weakly acid solution.
[0204] In the present invention, when Fmoc-protected amino acids or
Fmoc-protected amino acid analogs have protecting groups on their
side chains, preferable examples of such side-chain protecting
groups are as described below.
[0205] When the side-chain protecting groups are those for the
hydroxyl groups of Ser, Thr, Hyp, and derivatives thereof,
protecting groups having a MOM skeleton, Bn skeleton, Dpm skeleton,
Trt skeleton, silyl skeleton, or Boc skeleton each of which is
represented by the following general formulae are preferred.
##STR00009##
[0206] Representative examples of protecting groups having a MOM
skeleton include MOM (R1=H, R2=H, X=Me), EE (R1=Me, R2=H, X=Et),
MIP (R1=Me, R2=Me, X=Me), THP (R2=H, having a cyclic structure of
four carbon atoms by R1 and X), THF (R2=H, having a cyclic
structure of three carbon atoms by R1 and X), and SEM (R1=H, R2=H,
X=2-trimethylsilylethyl). Regarding the Me and Et substituents on
the skeleton, the skeleton substituted with other substituents such
as alkyl groups, benzyl groups, and aryl groups may be used.
##STR00010##
[0207] Representative examples of protecting groups having a Bn
skeleton include Pis (R6=Me, R7=Me, other Rs=H), PMB (R3=OMe, other
Rs=H), and DMB (R1=OMe, R3=OMe, other Rs=H). Instead of the Me
substituent group, another alkyl group may be used. Furthermore,
the benzene ring may have substituents such as alkyl groups, aryl
groups, and halogen groups.
##STR00011##
[0208] Representative examples of protecting groups having a Dpm
skeleton include Dpm (all Rs=H). The aromatic ring may have
substituents such as alkyl groups, aryl groups, alkoxy groups, and
halogen groups.
[0209] Groups in which R5 and R6 are bridged, such as the Xan group
in which R5 and R6 are bridged through an oxygen atom or a
dibenzosuberyl group in which R5 and R6 are bridged through two
carbon atoms may also be used.
##STR00012##
[0210] Representative examples of protecting groups having a Trt
skeleton include Trt (all Rs=H), Mmt (R3=Me, other Rs=H), Mtt
(R3=OMe, other Rs=H), Dmt (R3=OMe, R8=OMe, other Rs=H), and Clt
(R1=Cl, other Rs=H). The aromatic rings may have substituents such
as alkyl groups, aryl groups, alkoxy groups, and halogen
groups.
[0211] Furthermore, groups in which R5 and R6 are bridged, such as
the Pixel group in which R5 and R6 are bridged through an oxygen
atom may also be used.
##STR00013##
[0212] Representative examples of protecting groups having a silyl
skeleton include TBS (R1=Me, R2=Me, R3=tBu). Instead of Me and tBu,
other groups such as alkyl groups and aryl groups may be the
substituents.
##STR00014##
[0213] Representative examples of protecting groups having a Boc
skeleton include Boc (all s=H), but it may be substituted with
other alkyl groups, aryl groups, and such.
[0214] Additionally, protecting groups shown below may also be
used.
##STR00015##
[0215] Among these protecting groups, THP and Trt are particularly
preferred. Furthermore, THP and Trt are particularly preferred as
the side-chain protecting groups when the amino acid residue is
Ser, and THP is particularly preferred as the side-chain protecting
group when the amino acid residue is Thr.
[0216] When the side-chain protecting groups are those for amino
acids having an aryl group with a hydroxyl group substituent, such
as Tyr, D-Tyr, or Tyr(3-F), for example, protecting groups having a
MOM skeleton, Bn skeleton, Dpm skeleton, Trt skeleton, silyl
skeleton, Boc skeleton, or tBu skeleton which are represented by
the following general formula are preferred.
##STR00016##
[0217] Representative examples of protecting groups having a MOM
skeleton include MOM (R1=H, R2=H, X=Me), BOM (R1=H, R2=H, X=Bn), EE
(R1=Me, R2=H, X=Et), THP (R2=H, having a cyclic structure of four
carbon atoms by R1 and X), THF (R2=H, having a cyclic structure of
three carbon atoms by R1 and X), and SEM (R1=H, R2=H,
X=2-trimethylsilylethyl). Regarding the Me and Et substituent
groups on the skeletons, a skeleton substituted with other
substituents such as alkyl groups, benzyl groups, or aryl groups
may also be used.
##STR00017##
[0218] Representative examples of protecting groups having a Bn
skeleton include Pis (R6=Me, R7=Me, other Rs=H), PMB (R3=OMe, other
Rs=H), and DMB (R1=OMe, R3=OMe, other Rs=H). Instead of the Me
substituent group, other alkyl groups may be used. Furthermore, the
benzene ring may have substituents such as alkyl groups, aryl
groups, and halogen groups.
##STR00018##
[0219] Representative examples of protecting groups having a Dpm
skeleton include Dpm (all Rs=H). The aromatic ring may have
substituents such as alkyl groups, aryl groups, alkoxy groups, and
halogen groups.
[0220] Groups in which R5 and R6 are bridged, such as the Xan group
in which R5 and R6 are bridged through an oxygen atom or a
dibenzosuberyl group in which R5 and R6 are bridged through two
carbon atoms may also be used.
##STR00019##
[0221] Representative examples of protecting groups having a Trt
skeleton include Trt (all Rs=H), Mmt (R3=Me, other Rs=H), Mtt
(R3=OMe, other Rs=H), and Clt (R1=Cl, other Rs=H). The aromatic
ring may have substituents such as alkyl groups, aryl groups,
alkoxy groups, and halogen groups.
[0222] Furthermore, groups in which R5 and R6 are bridged, such as
the Pixel group in which R5 and R6 are bridged through an oxygen
atom may also be used.
##STR00020##
[0223] Representative examples of protecting groups having a silyl
skeleton include TBS (R1=Me, R2=Me, R3=tBu). Instead of the Me and
tBu, other groups such as alkyl groups and aryl groups may be the
substituents.
##STR00021##
[0224] Representative examples of protecting groups having a Boc
skeleton include Boc (all Rs=H), but it may be substituted with
other groups such as alkyl groups, aryl groups.
##STR00022##
[0225] Representative examples of protecting groups having a tBu
skeleton include tBu (all Rs=H). Instead of H, it may have
substituents such as alkyl groups and aryl groups.
[0226] Among these protecting groups, tBu, Pis, Trt, Ch, THP, and
THF are particularly preferred. Furthermore, when the amino acid
residue is Tyr or D-Tyr, the side-chain protecting group is
particularly preferably tBu, Trt, Clt, or THP, and when the amino
acid residue is Tyr(3-F), the side-chain protecting group is
particularly preferably tBu or Pis.
[0227] When the side-chain protecting groups are those for an amino
acid that has an imidazole on its side chain, such as His or MeHis,
use of protecting groups having, for example, a MOM skeleton, Bn
skeleton, or Trt skeleton which are represented by the following
general formulae is preferred.
##STR00023##
[0228] Representative examples of protecting groups having a MOM
skeleton include MBom (R1=H, R2=H, X=4-methoxybenzyl), 2,4-DMBom
(R1=H, R2=H, X=2,4-dimethoxybenzyl), 3,4-DMBom (R1=H, R2=H,
X=3,4-dimethoxybenzyl), EE (R1=Me, R2=H, X=Et), THP (R2=H, having a
cyclic structure of four carbon atoms by R1 and X), and THF (R2=H,
having a cyclic structure of three carbon atoms by R1 and X).
Regarding the Me and Et substituents on the skeleton, the skeleton
having protecting groups substituted with other substituents such
as alkyl groups, benzyl groups, or aryl groups may also be
used.
##STR00024##
[0229] Representative examples of protecting groups having a Bn
skeleton include Pis (R6=Me, R7=Me, other Rs=H), PMB (R3=OMe, other
Rs=H), and DMB (R1=OMe, R3=OMe, other Rs=H). Instead of the Me
substituent group, other alkyl groups may be used. Furthermore, the
benzene ring may have substituents such as alkyl groups, aryl
groups, and halogen groups.
##STR00025##
[0230] Representative examples of protecting groups having a Trt
skeleton include Trt (all Rs=H), Mmt (R3=Me, other Rs=H), Mtt
(R3=OMe, other Rs=H), and Clt (R1=Cl, other Rs=H). The aromatic
ring may have substituents such as alkyl groups, aryl groups,
alkoxy groups, and halogen groups.
[0231] Among them, Trt is particularly preferred. Furthermore, when
the amino acid residue is His or MeHis, the side-chain protecting
group is particularly preferably Trt.
[0232] Furthermore, protecting groups having a MOM skeleton, Bn
skeleton, Dpm skeleton, Trt skeleton, tBu skeleton, or phenyl-EDOTn
skeleton represented by the following general formulae can be used,
for example, as the protecting group for the side-chain carboxylic
acid group of Asp, Glu, and derivatives thereof when the main-chain
carboxylic acid group is used as a "free carboxylic acid group or
active esterifed carboxylic acid group", or as the protecting group
for the main-chain carboxylic acid group when the side-chain
carboxylic acid group of Asp, Glu, and derivatives thereof as a
"free carboxylic acid group or active esterified carboxylic acid
group". Furthermore, protecting groups having an orthoester
skeleton in which three alkoxy groups are bonded to a carboxylic
acid group-derived carbon atom can also be used as protecting
groups for carboxylic acids. Carbon atoms forming such protecting
groups may have substitutions.
##STR00026##
[0233] Representative examples of protecting groups having a MOM
skeleton include BOM (R1=H, R2=H, X=Bn), THP (R2=H, having a cyclic
structure of four carbon atoms by R1 and X), and THF (R2=H, having
a cyclic structure of three carbon atoms by R1 and X). For the
substituent groups on the skeleton, a skeleton having other
substituents such as alkyl groups, benzyl groups, or aryl groups
may also be used.
##STR00027##
[0234] Representative examples of protecting groups having a Bn
skeleton include Pis (R6=Me, R7=Me, other Rs=H), PMB (R3=OMe, other
Rs=H), DMB (R1=OMe, R3=OMe, other Rs=H), and piperonyl (R2 and R3
are both substituted with oxygen atoms, and those oxygen atoms are
bridged through a single carbon atom; other Rs=H). Instead of the
Me substituent group, other alkyl groups may be used. Furthermore,
the benzene ring may have substituents such as alkyl groups, aryl
groups, and halogen groups.
##STR00028##
[0235] Representative examples of protecting groups having a Dpm
skeleton include Dpm (all Rs=H). The aromatic ring may have
substituents such as alkyl groups, aryl groups, alkoxy groups, and
halogen groups.
[0236] Furthermore, groups in which R5 and R6 are bridged, such as
a dibenzosuberyl group in which R5 and R6 are bridged through two
carbon atoms may also be used.
##STR00029##
[0237] Representative examples of protecting groups having a Trt
skeleton include Trt (all Rs=H), Mint (R3=Me, other Rs=H), Mtt
(R3=OMe, other Rs=H), and Clt (R1=Cl, other Rs=H). The aromatic
ring may have substituents such as alkyl groups, aryl groups,
alkoxy groups, and halogen groups.
[0238] Furthermore, groups in which R5 and R6 are bridged, such as
the Pixyl group in which R5 and R6 are bridged through an oxygen
atom may also be used.
##STR00030##
[0239] Representative examples of protecting groups having a tBu
skeleton include tBu (all Rs=H), and Mpe (R1=Me, R4=Me, other
Rs=H). It may have substituents such as other alkyl groups and aryl
groups.
##STR00031##
[0240] Phenyl-EDOTn having the following combination of substituent
groups can be used: (i) R1=R2=R3=OMe; (ii) R1=R2=OMe, R3=H; (iii)
R1=R2=R3=OMe; or (iv) R1=R2=R3=H.
##STR00032##
[0241] A dicyclopropylmethyl group may also be used.
[0242] Among them, tBu, Pis, and Trt are particularly
preferred.
[0243] In the present invention, "Fmoc-protected peptide" means a
peptide comprising either one or both of the aforementioned
"Fmoc-protected amino acid" and "Fmoc-protected amino acid analog".
Examples of such peptides include dipeptides and oligopeptides
comprising a total of two or more molecules including either one or
both of the aforementioned Fmoc-protected amino acids and
Fmoc-protected amino acid analogs.
[0244] In peptide synthesis by the solid-phase methods of the
present invention, Fmoc-protected amino acids, Fmoc-protected amino
acid analogs, or Fmoc-protected peptides (also referred to as
Fmoc-protected amino acids and the can be supported onto a solid
phase using resins. The groups in the employed resin, which are
used for bonding to the Fmoc-protected amino acids and the like
(resin bonding group) are not particularly limited as long as they
allow peptides to be cleaved off by acids. The supported amount and
the supported ratio of the Fmoc-protected amino acids and the like
are not particularly limited either. In the present invention, for
example, a tritylchloride resin (Trt resin), a
2-chlorotritylchloride resin (Clt resin), a 4-methyltritylchloride
resin (Mtt resin), and 4-methoxytritylchloride resin (Mmt) can be
used. It is particularly preferred that the resins have resin
bonding groups which are described and have been evaluated to be "H
(<5% TFA in DCM)" as acid sensitivity in the Solid-phase
Synthesis Handbook (published by Merck Co. on May 1, 2002), and
they can be selected appropriately according to the functional
groups on the amino acids to be used. For example, when using a
carboxylic acid (main-chain carboxylic acid or side-chain
carboxylic acid represented by Asp or Glu) or a hydroxy group on an
aromatic ring (phenol group represented by Tyr) as the functional
group on the amino acid, use of trityl chloride resin (Trt resin)
or 2-chlorotritylchloride resin (Clt resin) as the resin is
preferred. When using an aliphatic hydroxy group (aliphatic alcohol
group represented by Ser or Thr) as the functional group on the
amino acid, use of tritylchloride resin (Trt resin),
2-chlorotritylchloride resin (Clt resin), or 4-methyltritylchloride
resin (Mtt) as the resin is preferred.
[0245] Furthermore, the types of polymers constituting the resins
are also not particularly limited. For resins composed of
polystyrenes, either 100 to 200 mesh or 200 to 400 mesh may be
used. The cross-link percentage is also not particularly limited,
but those cross-linked with 1% divinylbenzene (DVB) are
preferred.
[0246] Fmoc-protected amino acids, Fmoc-protected amino acid
analogs, or Fmoc-protected peptides are supported onto resins by
performing chemical reactions between the bonding groups on the
resins and the free carboxylic acid groups or the active esterified
carboxylic acid groups of Fmoc-protected amino acids, or
Fmoc-protected amino acid analogs, or amino acids positioned at the
C terminus of the Fmoc-protected peptides. In this case, the free
carboxylic acid may be the main-chain carboxylic acid of the amino
acids or amino acid analogs, or the side-chain carboxylic acids
(Asp and such). Instead of the carboxylic acid groups, free OH
groups or free SH groups of the side chains or main chains of
Fmoc-protected amino acids, Fmoc-protected amino acid analogs, or
amino acids positioned at the C-terminus of the Fmoc-protected
peptides, may also be used for supporting onto the solid phase.
[0247] The protecting groups having an Fmoc skeleton, which are
carried by Fmoc-protected amino acids, Fmoc-protected amino acid
analogs, or Fmoc-protected peptides supported onto the solid phase,
are deprotected by bases to expose their amino groups. The bases
used here is not particularly limited, and deprotecting agents
generally used in peptide synthesis may be used (for example, Amino
Acid-Protecting Groups (Chem. Rev. 2009, 109, 2455-2504)). Examples
of such deprotecting agents are preferably secondary amines, bases
having an amidine skeleton, and bases having a guanidine skeleton.
Specific examples of the secondary amines include piperidine,
morpholine, pyrrolidine, and piperidine. Specific examples of bases
having an amidine skeleton include
1,8-diazabicyclo[5.4.0]undeca-7-en (DBU) and
1,5-diazabicyclo[4.3.0]-5-nonen (DBN). Specific examples of the
bases having a guanidine skeleton include
1,1,3,3-tetramethylguanidine.
[0248] The aforementioned exposed amino groups and the free or
active esterified carboxylic acid groups of newly added
Fmoc-protected amino acids, Fmoc-protected amino acid analogs, or
Fmoc-protected peptides are condensed to form peptide bonds.
[0249] The condensing agents used when condensing amino groups and
carboxylic acid groups are not particularly limited as long as they
can form amide bonds, and condensing agents generally used in
peptide synthesis are preferred (for example, Peptide Coupling
Reagents, More than a Letter Soup (Chem. Rev. 2011, 111,
6557-6602)). Specific examples of such condensing agents include
condensing agents having a carbodiimide skeleton. For example,
condensing agents having a carbodiimide skeleton can be used for
condensation reactions by combining them with hydroxy compounds
that can form active esters. Examples of the condensing agents
having a carbodiimide skeleton include
N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide
(DIC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (WSCIHCl) (see for example, the catalog of Watanabe
Chemical: Amino Acids and Chiral Building Blocks to New Medicine).
Examples of the hydroxy compounds that can form active esters
include 1-hydroxy-1H-benzotriazole (HOBt),
1-hydroxy-7-azabenzotriazole (HOAt), ethyl
2-cyano-2-(hydroxyimino)acetate (oxyma),
3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazin (HOOBt or HODhbt),
N-hydroxy-5-norbomene-2,3-dicarboximide (HONB),
2,3,4,5,6-pentafluorophenol (HOPfp), N-hydroxysuccinimide (HOSu),
and 6-chloro-1-hydroxy-1H-benzotriazole (Cl-HOBt) (see for example,
the catalog of Watanabe Chemical: Amino Acids and Chiral Building
Blocks to New Medicine:). Furthermore, salts having such skeletons
such as K-oxyma, which is the potassium salt of oxyma, can also be
used. Among them, HOBt, HOAt, oxyma, and HOOBt are particularly
preferred. Even among them, combined use of DIC and HOAt, or
combined use of DIC and oxyma are preferred. In addition, the
following agents can be used in combination in the condensation
reaction:
[0250] as phosphonium condensing agents and uronium condensing
agents, any one of:
O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HBTU);
O-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU);
N-[1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino(morpholino)]ur-
onium hexafluorophosphate (COMU);
O-[(ethoxycarbonyl)cyanomethylene-amino]-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HOTU);
O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TBTU);
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TATU);
1H-benzotriazol-1-yloxy-tri(pyrolidino)phosphonium
hexafluorophosphate (PyBOP);
1H-benzotriazol-1-yloxy-tris(dimethylamino)phosphonium
hexafluorophosphate (BOP); bromotri(pyrolidino)phosphonium
hexafluorophosphate (PyBroP); chlorotri(pyrolidino)phosphonium
hexafluorophosphate (PyCloP);
(7-azabenzotriazol-1-yloxy)tripyrrolid-inophosphonium
hexafluorophosphate (PyAOP); bromotris(dimethylamino)phosphonium
hexafluorophosphate (Brop);
3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT);
N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate
(TSTU); N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium
hexafluorophosphate (HSTU);
O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N,N,N',N'-tetrameth-
yluronium tetrafluoroborate (TDBTU); tetramethylthiuronium
S-(1-oxide-2-pyridyl)-N,N,N',N'-tetrafluoroborate (TOTT); and
O-(2-oxo-1(2H)pyridyl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TPTU), and any one of the bases,
N,N-diisopropylethylamine (DIPEA); triethylamine (TEA);
2,4,6-trimethylpyridine (2,4,6-colidine); and 2,6-dimethylpyridine
(2,6-lutidine). Combined use of HATU and DIPEA, or combined use of
COMU and DIPEA is particularly preferred. In addition,
N,N'-carbonyldiimidazole (CDI), 1,1'-carbonyl-di-(1,2,4-triazole)
(CDT), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
chloride (DMT-MM), propylphosphonic acid anhydride (T3P), and such
may also be used as condensing agents.
[0251] The production methods of the present invention further
comprises the steps of: [0252] deprotecting the protecting group
having an Fmoc skeleton of the added new Fmoc-protected amino acid,
the new Fmoc-protected amino acid analog, or the new Fmoc-protected
peptide, by using a base to expose its amino group; and [0253]
forming an amide bond by further adding a new Fmoc-protected amino
acid, a new Fmoc-protected amino acid analog, or a new
Fmoc-protected peptide.
[0254] These steps may be repeated once or several times. With the
methods of the present invention, repeating deprotection of the
protecting group having the Fmoc skeleton and the condensation
reaction with a next, new Fmoc-protected amino acid, new
Fmoc-protected amino acid analog, or new Fmoc-protected peptide, a
desired peptide sequence can be obtained.
[0255] When the present invention is performed by solid-phase
methods, the desired peptide once obtained is cleaved off from the
solid phase (cleavage step). Furthermore, structural conversion and
cyclization of the peptide can be carried out before the cleavage
step. In the present invention, the side chain functional groups
that have been protected by protecting groups may be deprotected or
may not be deprotected. at the time of cleavage, and only a part of
the protecting groups may be deprotected. Preferably, the cleavage
step is carried out while the side-chain functional groups are
still protected.
[0256] Specifically, the reaction conditions for the cleavage step
of the present invention are preferably weakly acidic conditions,
and particularly preferably conditions of weaker acidity than TFA.
Specifically, for such weak acids, acids showing an aqueous pKa
value higher than that of TFA are preferred. More specifically,
acids having the pKa value in the range of 0 to 15 are preferred,
and those having an aqueous pKa value in the range of 6 to 15 are
more preferred. Examples of acids having weaker acidity than TFA,
which are used in this step, include TFE and HFIP. Two or more weak
acids can be used in combination, such as TFE/acetic acid, in any
ratios. Furthermore, a solvent such as DCM, DCE, and water can be
mixed in any ratios. For such combinations of weak acids and
solvents, the combination of TFE and DCM is particularly preferred.
For the solutions used for cleavage, other organic solvents and
reagents (for example, DIPEA), and cation scavengers (for example,
triisopropylsilane) may be added.
[0257] When the cleavage step is performed before deprotecting the
side-chain protecting groups of the synthesized peptides, the weak
acid used for the cleavage is preferably an acid weaker than the
acid used for the deprotection reaction. In this case, two types of
acids having different acidities which are weaker than TFA are
prepared in advance, and the weaker acid of the two is used for the
cleavage.
[0258] When the cleavage step is performed after deprotecting the
side-chain protecting groups of the synthesized peptides, the weak
acids used for the cleavage are not particularly limited as long as
they are acids weaker than TFA.
[0259] In the step of deprotecting the side-chain protecting groups
of the present invention, desired deprotection reactions can be
performed selectively by reducing side reactions such as hydrolysis
and N- to O-acyl shift. Deprotection of the side-chain protecting
groups is preferably performed under conditions of weaker acidity
than TFA. The deprotection reaction can be performed at any
temperature, and performing the reaction at 0.degree. C. to
40.degree. C. is preferred. When deprotection is completed, or when
stopping the reaction during deprotection, bases such as ammonia
and primary amines to tertiary amines can be used. Furthermore,
basic heterocyclic compounds (for example, pyridine, imidazole, and
analogs thereof) such may also be used.
[0260] When further altering or modifying the peptides synthesized
by the production methods of the present invention, those steps can
be performed either before or after the cleavage step.
[0261] Peptides produced by the production methods of the present
invention may be a peptide comprising on its C-terminal side an
amino acid residue or an amino acid analogue residue having one
reactive site on a side chain, and on its N-terminal side an amino
acid residue, an amino acid analogue residue, or a carboxylic acid
analog having another reactive site. Such peptides can be produced,
for example, by selecting the raw material Fmoc-protected amino
acids, Fmoc-protected amino acid analogs, and Fmoc-protected
peptides so that an amino acid residue or an amino acid analogue
residue having one reactive site on its side chain is comprised at
the C-terminal side and an amino acid residue, an amino acid
analogue residue, or a carboxylic acid analog having another
reactive site is comprised at the N-terminal side.
[0262] This peptide can be cyclized by forming a bond between one
reactive site and another reactive site. Production methods of the
present invention can comprise such a cyclization step.
Specifically, the cyclization step can be performed based on the
description in WO 2013/100132.
[0263] When performing the cyclization step after the cleavage
step, a concentrated residue obtained under reduced pressure from
reacted solution of the cleavage step (cleavage solution) may be
used in the cyclization step, or the cleavage solution may be used
as is in the cyclization step.
[0264] In the present invention, "carboxylic acid analog" includes
compounds having both an amino group and a carboxyl group and
having three or more atoms between the two groups various
carboxylic acid derivatives which do not have amino groups;
peptides formed from two to four residues; and amino acids in which
the main chain amino group has been chemically modified by
formation of an amide bond or such with a carboxylic acid.
Furthermore, "carboxylic acid analog" may have a borate or borate
ester moiety which can be used for cyclization. Furthermore,
"carboxylic acid analog" may be carboxylic acids having a double
bonded portion or a triple bonded portion, or may be carboxylic
acids having a ketone or a halide. In these compounds, portions
other than the specified functional groups may be substituted, and
for example, such substituents can be selected from among alkyl
groups, aralkyl groups, aryl groups, cycloalkyl groups, heteroaryl
groups, alkenyl groups, alkynyl groups, and such (freely selected
substituents).
[0265] The cyclization step comprises the step of cyclizing by
forming, for example, an amide bond, a disulfide bond, an ether
bond, a thioether bond, an ester bond, a thioester bond, or a
carbon-carbon bond by the above-mentioned two reactive sites, but
is not limited thereto.
[0266] Cyclizations by amide bond formation are, for example,
cyclizations by forming an amide bond between a reactive site on
the N-terminal amino acid residue, N-terminal amino acid analogue
residue, or N-terminal carboxylic acid analog (a main-chain amino
group or an amino group present on the side chain) and a reactive
site on the amino acid residue or amino acid analog having one
carboxylic acid on its side chain. As the condensing agents for
these reactions, agents similar to those used in the
above-described peptide bonding may be used. Specifically, for
example, the side-chain carboxylic acid and the N-terminal
main-chain amino group, or the side-chain amino group and the
C-terminal main-chain carboxylic acid can be condensed by using the
combination of HATU and DIPEA or the combination of COMU and DIPEA.
In this case, it is preferable that the protecting group for the
carboxylic acid on the C-terminal side and the protecting group for
the carboxylic acid on the side chain which is subjected to
cyclization, or the protecting group for the main-chain amino group
on the N-terminal side and the protecting group for the amino group
on the side chain which is subjected to cyclization are selected by
considering their orthogonality. The preferred protecting groups in
this series of peptide syntheses are as described above.
[0267] Cyclization by carbon-carbon bond formation is, for example,
cyclization by forming a carbon-carbon bond between a reactive site
on the N-terminal amino acid residue, N-terminal amino acid
analogue residue, or N-terminal carboxylic acid analog, and a
reactive site on the amino acid residue or amino acid analog having
one reactive site on its side chain. Specifically, for example, by
selecting an alkenyl group as the reactive site on the N-terminal
amino acid residue, N-terminal amino acid analogue residue, or
N-terminal carboxylic acid analog, and selecting an alkenyl group
as the reactive site on the amino acid residue or amino acid
analogue residue having one reactive site on its side chain, a
cyclization reaction can be carried out by a transition
metal-catalyzed carbon-carbon bonding reaction. In this case,
examples of the transition metals used as the catalyst include
ruthenium, molybdenum, titanium, and tungsten. For example, when
using ruthenium, the cyclization reactions can be carried out by
metathesis reactions. Furthermore, the cyclization reactions can be
carried out by transition metal-catalyzed carbon-carbon bonding
reactions by employing the combination of arylhalide and boronic
acid or boronic acid analog as the combination of reactive site on
the N-terminal amino acid residue, N-terminal amino acid analogue
residue, or N-terminal carboxylic acid analog and the reactive site
on the amino acid residue or amino acid analogue residue having one
reactive site on its side chain. In this case, the transition
metals used as the catalyst include palladium, nickel, and iron.
For example, when using palladium, the cyclization reactions can be
carried out by the Suzuki reaction. Furthermore, the cyclization
reactions can be carried out by a transition metal-catalyzed
carbon-carbon bonding reaction by employing the combination of an
alkenyl group and an aryl halide or alkenyl halide as the
combination of reactive site on the N-terminal amino acid residue,
N-terminal amino acid analogue residue, or N-terminal carboxylic
acid analog, and the reactive site on the amino acid residue or
amino acid analogue residue having one reactive site on its side
chain. In this case, the transition metals used as the catalyst
include palladium and nickel. For example, when using palladium,
the cyclization reactions can be carried out by the Heck-type
chemical reactions. Furthermore, the cyclization reactions can be
carried out by transition metal-catalyzed carbon-carbon bonding
reactions by selecting the combination of an acetylene group and an
amyl halide or alkenyl halide as the combination of reactive site
on the N-terminal amino acid residue, N-terminal amino acid
analogue residue, or N-terminal carboxylic acid analog and the
reactive site on the amino acid residue or amino acid analogue
residue having one reactive site on its side chain. In this case,
the transition metals used as the catalyst include palladium,
copper, gold, and iron. For example, when using the combination of
palladium and copper, the cyclization reactions can be carried out
by the Sonogashira reaction.
[0268] In the present invention, the obtained products can be
purified as necessary. For example, general peptide purification
methods such as reverse-phase columns or molecular sieve columns
may be used. Furthermore, the obtained products can also be
purified by crystallization or solidification using appropriate
solvents. Concentration under reduced pressure before purification
is also possible.
[0269] All prior art references cited herein are incorporated by
reference into this description.
EXAMPLES
[0270] The present invention will be further illustrated with
reference to the following Examples but is not limited thereto.
[0271] The following abbreviations are used in the Examples. [0272]
DCM Dichloromethane [0273] DCE 1,2-Dichloroethane [0274] DMF
N,N-dimethylformamide [0275] DIC N,N'-diisopropylcarbodiimide
[0276] DIPEA N,N-diisopropylethylamine [0277] DBU
1,8-Diazabicyclo[5.4.0]-7-undecene [0278] NMP
N-Methyl-2-pyrrolidone [0279] FA Formic acid [0280] TFA
Trifluoroacetic acid [0281] TFE 2,2,2-Trifluoroethanol [0282] HFIP
1,1,1,3,3,3-Hexafluoroisopropyl alcohol [0283] HOAt
1-Hydroxy-7-azabenzotriazole [0284] HOBt 1-Hydroxybenzotriazole
[0285] WSCl.HCl 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride [0286] TBME t-Butyl methyl ether [0287] TIPS
Triisopropyl silane [0288] HATU
O-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate
[0289] The reaction solvents used for peptide synthesis and
solid-phase synthesis were those for peptide synthesis (purchased
from Watanabe Chemical Industries and Wako Pure Chemical
Industries). Examples include DCM, DMF, NMP, 2% DBU in DMF, and 20%
piperidine in DMF. Furthermore, for reactions to which water was
not added as solvent, dehydrated solvents, super-dehydrated
solvents, and anhydrous solvents (purchased from Kanto Chemical
Co., Wako Pure Chemical Industries, and such) were used.
[0290] Conditions for LCMS analyses owe as shown in Table 1.
TABLE-US-00001 TABLE 1 Column Column Analysis (I.D. .times. Length)
Gradient Flow Rate Temperature Condition Device (mm) Mobile Phase
(A/B) (mL/min) (.degree. C.) Wavelength SQDAA05 Acquity Ascentis
Express A) 10 mM AcONH H.sub.2O 95/5 => 0/100 (1.0 min) 1 35
210-400 nm UPLC/SQD C18 (2.1 .times. 50) B) MeOH 0/100 (0.4 min)
PDA total SQDAA50 Acquity Ascentis Express A) 10 mM AcONH H.sub.2O
50/50 => 0/100 (0.7 min) 1 35 210-400 nm UPLC/SQD C18 (2.1
.times. 50) B) MeOH 0/100 (0.7 min) PDA total SQDFA05 Acquity
Ascentis Express A) 0.1% A H.sub.2O 95/5 => 0/100 (1.0 min) 1 35
210-400 nm UPLC/SQD C18 (2.1 .times. 50) B) 0.1% FA MeCN 0/100 (0.4
min) PDA total SQDFA50 Acquity Ascentis Express A) 0.1% FA H.sub.2O
50/50 => 0/100 (0.7 min) 1 35 210-400 nm UPLC/SQD C18 (2.1
.times. 50) B) 0.1% FA MeCN 0/100 (0.7 min) PDA total indicates
data missing or illegible when filed
Example 1
Basic Synthetic Route for Cyclic Peptides Comprising N-Methyl Amino
Acids in their Sequences
[0291] Solid-phase synthesis by the Fmoc method was employed for
synthesizing cyclic peptides comprising N-methyl amino acids in
their sequences, and the synthesis was performed by the synthetic
route described in FIG. 1, which involves the following five-steps:
[0292] A) elongating the peptide from the N terminus of Asp
supported onto a 2-chlorotrityl resin through its side-chain
carboxylic acid, by an Fmoc method using a peptide synthesizer;
[0293] B) cleaving the peptide off from the 2-chlorotrityl resin;
[0294] C) cyclizing the cleaved peptide by amide bonding through
condensation of the side-chain carboxylic acid of Asp (open
circular unit) and the amino group at the N terminus of the peptide
chain (triangular unit); [0295] D) deprotecting the protecting
groups of side-chain functional groups included in the peptide
chain; and [0296] E) purifying the compound by fractionation
HPLC.
[0297] In the Example, unless otherwise particularly stated, the
cyclic peptides were synthesized based on this basic synthetic
route.
Fmoc-Amino Acids Used in Peptide Synthesis by a Peptide
Synthesizer
[0298] In the peptide syntheses described in the Examples, the
following Fmoc-amino acids were used for the synthesis by a peptide
synthesizer (the aforementioned step A).
[0299] Fmoc-Pro-OH, Fmoc-Thr(Trt)-OH, Fmoc-Ile-OH, Fmoc-Trp-OH,
Fmoc-D-Tyr(tBu)-OH, Fmoc-D-Tyr(Clt)-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH,
Fmoc-MePhe-OH, Fmoc-MeAla-OH, Fmoc-MeGly-OH, Fmoc-MeLeu-OH,
Fmoc-Phe(4-CF3)-OH, Fmoc-b-Ala-OH, Fmoc-b-MeAla-OH, Fmoc-Nle-OH,
Fmoc-Met(O2)-OH, Fmoc-Phe(3-Cl)--OH, Fmoc-MeVal, and Fmoc-Val-OH
were purchased from Watanabe Chemical Industries, Chempep Inc.,
Chem-Impex International Inc., or such.
[0300] Fmoc-MeSer(DMT)-OH, Fmoc-MePhe(3-Cl)--OH,
Fmoc-MeAla(4-Thz)-OH, Fmoc-Hyp(Et)-OH, and Fmoc-.gamma.EtAbu-OH,
Fmoc-nPrGly-OH were synthesized by methods described in the
literature (Document: WO 2013/100132 A1).
[0301] Fmoc-Ser(THP)-OH (Compound 1), Fmoc-Thr(THP)-OH (Compound
2), Fmoc-MeSer(THP)-OH (Compound 6), Fmoc-MeHis(Trt)-OH (Compound
7), Fmoc-D-Tyr(THP)-OH (Compound 8), Fmoc-D-Tyr(Pis)-OH (Compound
11), Fmoc-Tyr(3-F,tBu)-OH (Compound 13), Fmoc-MePhe(4-Cl)--OH
(Compound 16), and Fmoc-Tyr(3-F,Pis)-OH (Compound 22) were
synthesized as follows. These synthesized Fmoc-amino acids were
used not only for synthesizing peptides but also for examining
deprotection of protecting groups of side-chain functional groups
and protecting groups of C-terminal carboxylic acid groups.
Example 1-1
Synthesis of
(2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((tetrahydro-2H-pyra-
n-2-yl)oxy)propanoic acid (Compound 1, Fmoc-Ser(THP)-OH)
##STR00033##
[0303] Toluene (10 mL) was added to a mixture of
(2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-hydroxypropanoic
acid (Fmoc-Ser-OH; purchased from Watanabe Chemical Industries; 1.0
g, 3.06 mmol) and pyridinium p-toluenesulfonate (PPTS; 0.038 g,
0.153 mmol), and the moisture included was removed by
azeotropically distilling off toluene under reduced pressure.
Super-dehydrated tetrahydrofuran (THF, 6.1 mL) and
3,4-dihydro-2H-pyran (1.9 mL, 21.3 mmol) were added to the obtained
residue, and this was stirred under nitrogen atmosphere at
50.degree. C. for four hours. After confirming disappearance of the
raw materials by LCMS (SQDFA05), the mixture was cooled to
25.degree. C., and ethyl acetate (6 mL) was added. Next, saturated
aqueous sodium chloride solution (6 mL) was added to wash the
organic layer, and the aqueous layer was extracted using ethyl
acetate (6 mL). All of the obtained organic layers were mixed, and
this was further washed twice with saturated aqueous sodium
chloride solution (6 mL), The organic layer was dried over sodium
sulfate and the solvent was distilled off under reduced
pressure.
[0304] The obtained residue was dissolved in tetrahydrofuran (THF,
12.2 mL), and then 1.0 M phosphate buffer solution (12.2 mL;
adjusted to pH 8.0) was added. This mixture was stirred at
50.degree. C. for three hours. After cooling it to 25.degree. C.,
ethyl acetate (12.2 mL) was added, and the organic and aqueous
layers were separated. Ethyl acetate (12.2 mL) was added to the
aqueous layer for extraction, and then all of the obtained organic
layers were mixed, and this mixture was washed twice with saturated
aqueous sodium chloride solution (12.2 mL). The organic layer was
then dried over sodium sulfate, the solvent was distilled off under
reduced pressure, and the resulting material was further dried at
25.degree. C. for 30 minutes under reduced pressure using a
pump.
[0305] The obtained residue was dissolved in dichloromethane (7
mL), and then heptane (16.6 mL) was added. Under a controlled
reduced pressure (approximately 100 hPa), dichloromethane alone was
distilled off, and the obtained mixture was filtered to obtain
solid. This washing operation using heptane was repeated twice. The
obtained solid was dried at 25.degree. C. for two hours under
reduced pressure using a pump, and 1.40 g of residue was
obtained.
[0306] t-Butyl methyl ether (TBME, 25 mL) 0.05 M aqueous phosphoric
acid solution (70 mL; pH 2.1) were added to the obtained residue,
and after stirring this at 25.degree. C. for five minutes, the
organic and aqueous layers were separated. t-Butylmethyl ether
(TBME, 25 mL) was added to the aqueous layer for extraction, and
this was followed by mixing all of the obtained organic layers, and
washing this mixture twice with saturated aqueous sodium chloride
solution (25 mL). The organic layer was then dried over sodium
sulfate, and the solvent was distilled off under reduced pressure.
The residue was dried at 25.degree. C. for two hours under reduced
pressure using a pump to yield
(2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((tetrahydro-2H-pyra-
n-2-yl)oxy)propanoic acid (Compound 1, Fmoc-Ser(THP)-OH, 1.22 g, 30
mol % t-butyl methyl ether (TBME) remained). The obtained
Fmoc-Ser(THP)-OH was stored in a -25.degree. C. freezer. [0307]
LCMS (ESI) m/z=410.2 (M-H)- [0308] Retention time: 0.81 minutes
(analysis condition SQDFA05)
Example 1-2
Synthesis of
(2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((tetrahydro-2H-p-
yran-2-yl)oxy)butanoic acid (Compound 2, Fmoc-Thr(THP)-OH)
##STR00034##
[0310] Toluene (50 mL) was added to a mixture of
(2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-hydroxybutanoic
acid monohydrate (monohydrate of Fmoc-Thr-OH; purchased from Tokyo
Chemical Industry; 5.0 g, 13.9 mmol) and pyridinium
p-toluenesulfonate (PPTS; 0.175 g, 0.70 mmol), and the moisture
included was removed by azeotropically distilling off toluene under
reduced pressure. Super-dehydrated tetrahydrofuran (THF, 28 mL) and
3,4-dihydro-2H-pyran (8.8 mL, 97 mmol) were added to the obtained
residue, and this was stirred under nitrogen atmosphere at
50.degree. C. for four hours. After confirming disappearance of the
raw materials by LCMS (SQDFA05), the mixture was cooled to
25.degree. C., and ethyl acetate (30 mL) was added. Next, saturated
aqueous sodium chloride solution (30 mL) was added to wash the
organic layer, and the aqueous layer was extracted with ethyl
acetate (30 mL). All of the obtained organic layers were mixed, and
this was further washed twice with saturated aqueous sodium
chloride solution (30 mL). The organic layer was dried over sodium
sulfate and the solvent was distilled off under reduced pressure to
yield 9.3 g of a crude product.
[0311] 4.65 g of the obtained crude product was dissolved in
tetrahydrofuran (THF, 30 mL), and 1.0 M phosphate buffer solution
(30 mL; adjusted to pH 8.0) was added to it. This mixture was
stirred at 50.degree. C. for four hours. After cooling to
25.degree. C., ethyl acetate (30 mL) was added, and the organic and
aqueous layers were separated. After adding ethyl acetate (30 mL)
to the aqueous layer for extraction, all of the obtained organic
layers were mixed and washed twice with saturated aqueous sodium
chloride solution (30 mL). The organic layer was then dried over
sodium sulfate, the solvent was distilled off under reduced
pressure. This was further dried at 25.degree. C. for 30 minutes
under reduced pressure using a pump.
[0312] The obtained residue was dissolved in diethyl ether (50 mL),
and then heptane (50 ML) was added. Under a controlled reduced
pressure (approximately 100 hPa), diethyl ether alone was distilled
off, and the obtained mixture was filtered to yield a solid. This
washing operation using heptane was repeated twice. The obtained
solid was dried at 25.degree. C. for two hours under reduced
pressure using a pump, and the sodium salt of Fmoc-Thr(THP)-OH
(2.80 g, 6.26 mmol) was obtained.
[0313] To the total amount of the obtained sodium salt of
Fmoc-Thr(THP)-OH, ethyl acetate (50 mL) and 0.05 M aqueous
phosphoric acid solution (140 mL; pH 2.1) were added. After
stirring this at 25.degree. C. for five minutes, the organic and
aqueous layers were separated. Ethyl acetate (50 mL) was added to
the aqueous layer for extraction, and all of the obtained organic
layers were mixed and this mixture was washed twice with saturated
aqueous sodium chloride solution (50 mL). The organic layer was
then dried over sodium sulfate, and the solvent was distilled off
under reduced pressure. The residue was dried at 25.degree. C. for
two hours under reduced pressure using a pump, and then the
obtained solid was dissolved in t-butyl methyl ether (TBME, 50 mL),
and the solvent was distilled off under reduced pressure. This was
further dried at 25.degree. C. for one hour under reduced pressure
using a pump, and
(2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((tetrahydro-2H-p-
yran-2-yl)oxy)butanoic acid (Compound 2, Fmoc-Thr(THP)-OH, 2.70 g,
30 mol % t-butyl methyl ether (TBME) remained) was yielded as a
diastereomer formed due to the asymmetric carbon on the THP
protecting group. The obtained Fmoc-Thr(THP)-OH was stored in a
-25.degree. C. freezer. [0314] LCMS (ESI) m/z=424.2 (M-H)- [0315]
Retention time: 0.84 minutes, 0.85 minutes (analysis condition
SQDFA05)
Example 1-3
Synthesis of
(2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-((tetrahydro-
-2H-pyran-2-yl)oxy)propanoic acid (Compound 6,
Fmoc-MeSer(THP)-OH)
##STR00035##
[0317]
(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-hydroxy-
propanoic acid (Fmoc-MeSer-OH) was synthesized by a method
described in the literature (Document: WO 2013/100132 A1). To a
solution of
(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-hydroxypropan-
oic acid (Fmoc-MeSer-OH; 15 g, 43.9 mmol) in tetrahydrofuran (88
mL), pyridinium p-toluenesulfonate (PPTS; 0.552 g, 2.197 mmol) and
3,4-dihydro-2H-pyran (23.85 mL) were added, and this was stirred at
50.degree. C. for four hours. The mixture was cooled to 25.degree.
C., and ethyl acetate (90 mL) was added. Next, the organic layer
was washed with saturated aqueous sodium chloride solution (90 mL),
and the aqueous layer was extracted with ethyl acetate (90 mL). All
of the obtained organic layers were mixed, and this was further
washed twice with saturated aqueous sodium chloride solution (90
mL). The organic layer was dried over sodium sulfate, and the
solvent was distilled off under reduced pressure.
[0318] 15.0 g of the obtained residue was dissolved in
tetrahydrofuran (175 mL), and 1.0 M phosphate buffer (175 mL;
adjusted to pH 8.0) was added to it. This mixture was stirred at
50.degree. C. for three hours. After cooling to 25.degree. C.,
ethyl acetate (175 mL) was added, and the organic and aqueous
layers were separated. Ethyl acetate (175 mL) was added to the
aqueous layer for extraction, and all of the obtained organic
layers were mixed and washed twice with saturated aqueous sodium
chloride solution (175 mL). The organic layer was then dried over
sodium sulfate, and the solvent was distilled off under reduced
pressure.
[0319] The obtained residue was dissolved in dichloromethane (100
mL), and then heptane (250 mL)was added. Under a controlled reduced
pressure (approximately 100 hPa), dichloromethane alone was
distilled off, and the obtained mixture was filtered to yield a
solid. This washing operation using heptane was repeated twice. The
obtained solid was dried at 25.degree. C. for two hours under
reduced pressure using a pump.
[0320] t-Butyl methyl ether (TBME, 250 mL) and 0.05 M aqueous
phosphoric acid solution (700 mL; pH 2.1) were added to the
obtained residue. After stirring this at 25.degree. C. for five
minutes, the organic and aqueous layers were separated. t-Butyl
methyl ether (TBME, 250 mL) was added to the aqueous layer for
extraction, and all of the obtained organic layers were mixed and
washed twice with saturated aqueous sodium chloride solution (250
mL). The organic layer was then dried over sodium sulfate, and the
solvent was distilled off under reduced pressure. The residue was
dried at 25.degree. C. for two hours under reduced pressure using a
pump to yield
(2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-((t-
etrahydro-2H-pyran-2-yl)oxy)propanoic acid (Compound 6,
Fmoc-MeSer(THP)-OH, 9.0 g, 30 mol % t-butylmethyl ether (TBME)
remained). The obtained Fmoc-MeSer(THP)-OH was stored in a
-25.degree. C. freezer. [0321] LCMS (ESI) m/z=426.4 (M+H)+ [0322]
Retention time: 0.86 minutes (analysis condition SQDFA05)
Example 1-4
Synthesis of
(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-(1-trityl-1H--
imidazol-4-yl)propanoic acid (Compound 7, Fmoc-MeHis(Trt)-OH)
##STR00036##
[0324] A solution of
(S)-3-(1H-imidazol-4-yl)-2-(methylamino)propanoic acid
hydrochloride (75 g, 364.71 mmol) in dichloromethane (1000 mL),
dichlorodimethylsilane (51 g, 395.16 mmol), and triethylamine (40
g, 395.30 mmol) were added to a 3000-mL flask. Next a solution of
(chloromethanetrityl)tribenzene (Trt-Cl; 111 g, 398.17 mmol) in
dichloromethane (500 mL) and triethylamine (40 g, 395.30 mmol) were
added. The obtained reaction solution was stirred while heating
under reflux for four hours, and further stirred at 20.degree. C.
for two hours. The reaction was stopped by adding methanol to the
reaction solution, and then the solvent was distilled off under
reduced pressure. pH was adjusted to 8 to 8.5 using triethylamine
and 125 g of solid was obtained.
[0325] 1,4-Dioxane (1000 mL), potassium carbonate (84 g, 603.39
mmol) and water (1000 mL) were added to the obtained solid. In
addition, (2,5-dioxopyrrolidin-1-yl) (9H-fluoren-9-yl)methyl
carbonate (Fmoc-OSu; 102 g, 302.38 mmol) was added, and the mixture
was stirred at 0.degree. C. for two hours. The obtained reaction
solution was washed with diethyl ether (2000 mL), and then the pH
of the solution was adjusted to 6 to 7 using acetic acid. The
obtained solid was filtered to obtain
(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-(1-trityl-1H--
imidazol-4-yl)propanoic acid (Compound 7, Fmoc-MeHis(Trt)-OH, 155
g). [0326] LCMS (ESI) m/z=634.4 (M+H)+ [0327] Retention time: 1.07
minutes (analysis condition SQDAA05)
Example 1-5
Synthesis of
(2R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-((tetrahydro-2H-p-
yran-2-yl)oxy)phenyl)propanoic acid (Compound 8,
Fmoc-D-Tyr(THP)-OH)
##STR00037##
[0329] Toluene (5.0 mL) was added to
(R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino-3-(4-hydroxyphenyl)propa-
noic acid (Fmoc-D-Tyr-OH, purchased from Watanabe Chemical
Industries; 500 mg, 1.24 mmol) and a catalytic amount of pyridinium
para-toluene sulfonate (PPTS; 15.6 mg, 0.062 mmol), and the
included moisture was removed azeotropically by distilling off
toluene under reduced pressure. The obtained residue was dissolved
in tetrahydrofuran (THF) (2.5 mL), 3,4-dihydro-2H-pyran (785 .mu.L,
8.68 mmol) was added, and this was stirred under nitrogen
atmosphere at 50.degree. C. for four hours. The reaction solution
was cooled to 25.degree. C., and ethyl acetate (3 mL) was added.
Next, saturated aqueous sodium chloride solution (3 mL) was added
to wash the organic layer, and the aqueous layer was extracted with
ethyl acetate (3 mL). All of the obtained organic layers were
mixed, and this was further washed twice with saturated aqueous
sodium chloride solution (3 mL). The organic layer was dried over
sodium sulfate, the solvent was distilled off under reduced
pressure, and further the resulting material was dried under
reduced pressure using a pump, which yielded 596 mg of residue.
[0330] The obtained residue (300 mg) was dissolved in
tetrahydrofuran (THF) (2.5 mL), and then 1.0 M aqueous phosphoric
acid solution (pH 8.0, 2.5 mL) was added and stirred at 50.degree.
C. for three hours. Ethyl acetate (3 mL) was added to the reaction
solution, the organic and aqueous layers were separated, and the
aqueous layer was extracted with ethyl acetate (3 mL). All of the
obtained organic layers were mixed, and this was washed twice with
saturated aqueous sodium chloride solution (3 mL). The organic
layer was dried over sodium sulfate, then the solvent was distilled
off under reduced pressure, and the resulting material was further
dried for 30 minutes under reduced pressure using a pump.
[0331] The obtained residue was dissolved in dichloromethane (DCM)
(2 mL), and then heptane (5 mL) was added. Dichloromethane (DCM)
alone was removed using an evaporator, and the obtained white solid
was collected by filtration. Similar operations were repeated twice
on the obtained white solid. White solid obtained in this manner
was dried under reduced pressure using a pump for two hours.
[0332] To the above white solid, t-butyl methyl ether (TBME) (4.6
mL) and 0.05 M aqueous phosphoric acid solution (pH 2.1, 13 mL)
were added, and this was stirred at 25.degree. C. for five minutes.
After separating the organic layer, the aqueous layer was extracted
with t-butyl methyl ether (TBME) (4.6 mL). The obtained organic
layers were combined, and this was washed twice with saturated
aqueous sodium chloride solution (4.6 mL). The organic layer was
dried over sodium sulfate, and the solvent was distilled off under
reduced pressure. The obtained residue was purified by
reverse-phase chromatography (Wakosil 25C18, 10 g,
water/acetonitrile) to obtain
(2R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-((tetra-
hydro-2H-pyran-2-yl)oxy)phenyl)propanoic acid (Compound 8,
Fmoc-D-Tyr(THP)-OH, 173 mg) in 57% yield as a diastereomer formed
due to the asymmetric carbon on the THP protecting group. [0333]
LCMS (ESI) m/z=488.4 (M+H)+ [0334] Retention time: 0.92 minutes
(analysis condition SQDFA05)
Example 1-6
Synthesis of 2-phenylpropan-2-yl 2,2,2-trichloroacetoimidate
(Compound 9)
##STR00038##
[0336] A 1.9 M solution of NaHMDS in tetrahydrofuran (THF) (850
.mu.L, 1.62 mmol) was added dropwise at 22.degree. C. to a solution
of 2-phenylpropan-2-ol (purchased from Wako Pure Chemical
Industries; 2.0 g, 14.7 mmol) in diethyl ether (Et2O) (4.8 mL). The
reaction solution was stirred at the same temperature for 20
minutes, then cooled to 0.degree. C., and
2,2,2-trichloroacetonitrile (1.47 mL, 14.7 mmol) was added
dropwise. After stirring the reaction solution at 0.degree. C. for
ten minutes, the temperature was raised to 15.degree. C., and this
was stirred for another hour. The reaction solution was
concentrated using an evaporator, hexane (1.8 mL) and methanol (65
.mu.L) were added to the obtained residue, and this was stirred at
15.degree. C. for 15 minutes. The obtained solids were filtered and
washed three times with hexane (2.0 mL) to obtain 4.19 g of
2-phenylpropan-2-yl 2,2,2-trichloroacetoimidate (Compound 9). This
was used in reactions without further purification.
[0337] 1H NMR (Varian 400-MR, 400 MHz, CDCl3) .delta. 1.89 (6H, s),
7.28 (1H, m), 7.36 (2H, m), 7.43 (2H, m), 8.20 brs)
Example 1-7
Synthesis of (R)-methyl
2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-hydroxyphenyl)propanoa-
te (Compound 10, Fmoc-D-Tyr-OMe)
##STR00039##
[0339] Under nitrogen atmosphere, thionyl chloride (1.59 mL, 21.76
mmol) was added dropwise at 0.degree. C. to a mixture of
(R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(tert-butoxy)pheny-
l)propanoic acid (Fmoc-D-Tyr(tBu)-OH, purchased from Watanabe
Chemical Industries; 5.0 g, 10.88 mmol) and methanol (8.80 mL, 218
mmol). The obtained reaction solution was stirred at 25.degree. C.
for three hours, and then the solvent was distilled off under
reduced pressure. The obtained residue was dissolved in ethyl
acetate, and this solution was washed twice with saturated aqueous
sodium chloride solution. The organic layer was dried over sodium
sulfate, solids were removed by filtration, and the solvent was
distilled off under reduced pressure to obtain (R)-methyl
2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-hydroxyphenyl)propanoa-
te (Compound 10, Fmoc-D-Tyr-OMe, 4.50 g). [0340] LCMS (ESI)
m/z=418.3 (M+H)+ [0341] Retention time: 0.81 minutes (analysis
condition SQDFA05)
Example 1-8
Synthesis of
(R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-((2-phenylpropan-2-
-yl)oxy)phenyl)propanoic acid (Compound 11, Fmoc-D-Tyr(Pis)-OH)
##STR00040##
[0343] To a solution of (R)-methyl
2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-hydroxyphenyl)propanoa-
te (Compound 10, Fmoc-D-Tyr-OMe; 100 mg, 0.24 mmol) in
tetrahydrofuran (THF) (240 .mu.L), a separately prepared 10 M
solution of 2-phenylpropan-2-yl 2,2,2-trichloroacetoimidate
(Compound 9) in cyclohexane (60 .mu.L) and a catalytic amount of
boron trifluoride-ethyl ether complex (BF3-OEt, 4.55 .mu.L, 0.036
mmol) was added dropwise at 0.degree. C. After stirring the
reaction solution at 25.degree. C. for one hour, the equivalent
amount of the 10 M solution of 2-phenylpropan-2-yl
2,2,2-trichloroacetoimidate (Compound 9) in cyclohexane (60 .mu.L)
and boron trifluoride-ethyl ether complex (BF3-OEt, 455 .mu.L,
0.036 mmol) were added again, and this reaction solution was
stirred at 25.degree. C. for another 30 minutes. The reaction
solution was diluted with dichloromethane (DCM), and a saturated
aqueous sodium bicarbonate solution was added. After extraction
with dichloromethane, the organic layer was washed with saturated
aqueous sodium chloride solution. This organic layer was dried over
sodium sulfate, the solvent was distilled off under reduced
pressure, and the resulting material was further dried using a
pump. A solution of dichloromethane (DCM)/hexane=1/1 was added to
the obtained residue, and precipitates were removed by filtration.
The filtrate was concentrated using an evaporator, the obtained
residue was purified by flash column chromatography (purif pack
(registered trademark) SIZE 20, hexane/ethyl acetate), and
(R)-methyl
2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-((2-phenylpropan-2-yl)-
oxy)phenyl)propanoate (Fmoc-D-Tyr(Pis)-OMe) was obtained as a
mixture.
[0344] The obtained mixture described above was dissolved in
dichloroethane (DCE) (535 .mu.L), trimethyltin(IV) hydroxide
(Me3SnOH, 58.1 mg, 0.321 mmol) was added, and this was stirred at
60.degree. C. for seven hours. Trimethyltin(IV) hydroxide (Me3SnOH,
29.1 mg, 0.161 mmol) was further added to the reaction solution,
and this was stirred at 60.degree. C. for 15 hours. The reaction
solution was concentrated using an evaporator, t-butyl methyl ether
(TBME, 1 mL) and 0.05 M aqueous phosphoric acid solution (pH 21, 2
mL) were added, and this was stirred at 25.degree. C. for five
minutes. After separating the organic layer, the aqueous layer was
extracted twice with t-butyl methyl ether (TBME, 1 mL). The organic
layer was dried over sodium sulfate, the solvent was distilled off
under reduced pressure, and the resulting material was further
dried using a pump. The obtained residue was purified by column
chromatography (purif pack (registered trademark) SIZE 20,
dichloromethane/methanol), and
(R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-((2-phenylpropan-2-
-yl)oxy)phenyl)propanoic acid (Compound 11, Fmoc-D-Tyr(Pis)-OH, 33
mg) was obtained in a two-step yield of 39%. [0345] LCMS (ESI)
m/z=522.4 (M+H)+ [0346] Retention time: 1.00 minutes (analysis
condition SQDFA05)
Example 1-9
Synthesis of (S)-methyl
2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-hydroxyphenyl-
)propanoate (Compound 12, Fmoc-Tyr(3-F)-OMe)
##STR00041##
[0348] (S)-2-amino-3-(3-fluoro-4-hydroxyphenyl)propanoic acid
(H2N-Tyr(3-F)-OH, purchased from Astatech; 2.0 g, 10.0 mmol) was
dissolved in 10% aqueous sodium carbonate solution, and then, using
a dropping funnel, a solution of (2,5-dioxopyrrolidin-1-yl)
(9H-fluoren-9-yl)methyl carbonate (Fmoc-OSu, 3.39 g, 10.0 mmol) in
1,4-dioxane (35 mL) added at 0.degree. C. After stirring the
reaction solution at 25.degree. C. for 40 minutes, water (35 mL)
and diethyl ether (70 mL) were added, and the reaction solution was
washed three times with diethyl ether. The pH of the aqueous layer
was adjusted to 2 to 3 using an aqueous 5N hydrochloric acid
solution, and then this was extracted three times with ethyl
acetate (100 mL.times.3). The organic layer was dried over
magnesium sulfate, the solvent was distilled off under reduced
pressure, and the resulting material was further dried using a
pump. The obtained residue (4.08 g) was used in the next reaction
without further purification.
[0349] The above residue (1.04 g) was dissolved in methanol (10
mL), and thionyl chloride (SOCl2, 539 .mu.L, 7.38 mmol) was added
dropwise at 0.degree. C. The reaction solution was stirred at
60.degree. C. for one hour and then cooled to room temperature, and
the solvent was distilled off using an evaporator. Ethyl acetate
and water were added to the obtained residue, and extraction was
performed twice using ethyl acetate. The organic layer was washed
with saturated aqueous sodium chloride solution and dried over
magnesium sulfate, and the solvent was distilled off under reduced
pressure and further dried using a pump. The obtained residue was
purified by flash column chromatography (purif pack (registered
trademark) SIZE 200, hexane/ethyl acetate), and (S)-methyl
2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-hydroxyphenyl-
)propanoate (Compound 12, Fmoc-Tyr(3-F)-OMe; 900 mg, 2.07 mmol) was
obtained in a two-step yield of 84%. [0350] LCMS (ESI) m/z=436.4
(M+H)+ [0351] Retention time: 0.82 minutes (analysis condition
SQDFA05)
Example 1-10
Synthesis of
(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(tert-butoxy)-3-fl-
uorophenyl)propanoic acid (Compound 13, Fmoc-Tyr(3-F,tBu)-OH)
##STR00042##
[0353] To a solution of (S)-methyl
2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-hydroxyphenyl-
)propanoate (Compound 12, Fmoc-Tyr(3-F)-OMe; 300 mg, 0.689 mmol) in
tetrahydrofuran (THF) (690 .mu.L), tert-butyl
2,2,2-trichloroacetimidate (308 .mu.L, 1.72 mmol) and a catalytic
amount of boron trifluoride-ethyl ether complex (BF3-OEt, 13.1
.mu.L, 0.103 mmol) was added dropwise at 0.degree. C. After
stirring the reaction solution at 25.degree. C. for one hour, the
equivalent amount of tert-butyl 2,2,2-trichloroacetoimidate (308
.mu.L, 1.72 mmol) and boron trifluoride-ethyl ether complex
(BF3-OEt, 13.1 .mu.L, 0.103 mmol) were added again, and this
reaction solution was stirred at 25.degree. C. for another hour.
The reaction solution was diluted with dichloromethane (DCM), and a
saturated aqueous sodium bicarbonate solution was added. After
extraction with dichloromethane, the organic layer was washed with
saturated aqueous sodium bicarbonate solution and saturated aqueous
sodium chloride solution. The organic layer was dried over sodium
sulfate, the solvent was distilled off under reduced pressure, and
the resulting material was further dried using a pump. The obtained
residue was purified by flash column chromatography (purif pack
(registered trademark) SIZE 60, hexane/ethyl acetate), and
(S)-methyl
2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(tert-butoxy)-3-fluoro-
phenyl)propanoate (Fmoc-Tyr(3-F,tBu)-OMe) was obtained as a
mixture.
[0354] The obtained mixture (40 mg) described above was dissolved
in dichloroethane (DCE) (810 .mu.L), trimethyltin(IV) hydroxide
(Me3SnOH, 29.4 mg, 0.163 mmol) was added to this, and the mixture
was stirred at 60.degree. C. for one hour. After adding formic acid
(15.35 .mu.L, 0.407 mmol) to the reaction solution, this was
purified by reverse-phase chromatography (Wakosil 25C18, 10 g, 0.1%
aqueous formic acid solution/0.1% solution of formic acid in
acetonitrile) to obtain
(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(tert-butoxy)-3-fl-
uoropheny)propanoic acid (Compound 13, Fmoc-Tyr(3-F,tBu)-OH; 27 mg,
56.5 .mu.mol) in a two-step yield of 93%. [0355] LCMS (ESI)
m/z=478.3 (M+H)+ [0356] Retention time: 0.94 minutes (analysis
condition SQDFA05)
Example 1-11
Synthesis of 2-(2-phenylpropan-2-yl)
(S)-1-((9H-fluoren-9-yl)methyl) pyrrolidine-1,2-dicarboxylate
(Compound 14, Fmoc-Pro-OPis)
##STR00043##
[0358] To a mixture of 2-phenyl-2-propanol (14.2 g, 104 mmol) and
dehydrated diethyl ether (35 mL), 1.9 M NaHMDS (solution in
tetrahydrofuran; 0.85 mL, 1.62 mmol) was added dropwise over a
period of three minutes or more under nitrogen atmosphere at room
temperature, and then this was stirred at room temperature for 30
minutes.
[0359] Subsequently, the reaction solution was cooled on ice to
0.degree. C., and trichloroacetonitrile (11.5 mL, 115 mmol.) was
added dropwise over a period of five minutes or more. The mixture
was stirred at 0.degree. C. for ten minutes, then removed from its
ice-bath, and was further stirred at room temperature for one hour.
The obtained mixture was cooled on ice to 0.degree. C. and a
mixture of
(S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)pyrrolidine-2-carboxylic
acid (Fmoc-Pro-OH; 42.3 g, 12.5 mmol) and dichloromethane (100 mL)
was added over a period of 15 minutes. This was stirred at
0.degree. C. for 30 minutes, then filtered, and washed with a
hexane-dichloromethane (5/1) solution. Then, the solvent was
distilled off under reduced pressure. The obtained residue was
purified by silica gel column chromatography (hexane-ethyl acetate)
to yield 2-(2-phenylpropan-2-yl) (S)-1-((9H-fluoren-9-yl)methyl)
pyrrolidine-1,2-dicarboxylate (Compound 14, Fmoc-Pro-OPis; 26.3 g,
57.7 mmol). [0360] LCMS (ESI) m/z=456.4 (M+H)+ [0361] Retention
time: 0.76 minutes (analysis condition SQDAA50)
Example 1-12
Synthesis of
(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-(4-chlorophen-
yl)propanoic acid (Compound 16, Fmoc-MePhe(4-Cl)--OH)
##STR00044##
[0363] To a solution of
(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-chlorophenyl)propa-
noic acid (Fmoc-Phe(4-Cl)--OH; 170 g, 402.96 mmol) in toluene (2.5
L), paraformaldehyde (48 g, 1.60 mol) and 10-camphorsulfonic acid
(CSA; 4.6 g, 19.83 mmol) were added, and this was stirred at
110.degree. C. for 16 hours. Subsequently, the reaction solution
was washed twice with saturated aqueous sodium bicarbonate solution
(1 L), and twice with saturated aqueous sodium chloride solution (1
L). The organic layer was dried over sodium sulfate, solids were
removed by filtration, and the solvent was distilled off under
reduced pressure to obtain 160 g of (S)-(9H-fluoren-9-yl)methyl
4-(4-chlorobenzyl)-5-oxooxazoline-3-carbonate.
[0364] To a solution of (S)-(9H-fluoren-9-yl)methyl
4-(4-chlorobenzyl)-5-oxooxazoline-3-carbonate prepared by mixing a
separate lot prepared by similar operations (230 g, 530.10 mmol) in
dichloromethane (2.5 L), triethylsilane (881 g, 7.58 mol) and
trifluoroacetic acid (TFA; 2518 g, 22.28 mol) were mixed, and the
mixture was stirred at 30.degree. C. for 12 hours. Subsequently,
the solvent was distilled off under reduced pressure, and by
recrystallizing the obtained residue in dichloromethane/hexane
(1/10, v/v),
(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-(4-chlorophen-
yl)propanoic acid (Compound 16, Fmoc-MePhe(4-Cl)--OH, 205 g) was
obtained. [0365] LCMS (ESI) m/z=436.3 (M+H)+ [0366] Retention time:
0.99 minutes (analysis condition SQDAA05)
Example 1-13
Synthesis of (S)-methyl
2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-((2-phenylpro-
pan-2-yl)oxy)phenyl)propanoate (Compound 21,
Fmoc-Tyr(3-F,Pis)-OMe)
##STR00045##
[0368] (S)-methyl
2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-hydroxyphenyl-
)propanoate (Compound 12, Fmoc-Tyr(3F)-OMe; 200 mg, 0.459 mmol) was
dissolved in THF (460 .mu.L), and then a separately prepared
2-phenylpropan-2-yl 2,2,2-trichloroacetoimidate (Compound 9; 322
mg, 1.15 mmol) and a catalytic amount of boron trifluoride-ethyl
ether complex (BF3-OEt, 8.73 .mu.L, 0.069 mmol) were added dropwise
at 0.degree. C. After stirring the reaction solution at room
temperature for 30 minutes, the equivalent amount of
2-phenylpropan-2-yl 2,2.2-trichloroacetoimidate (322 mg, 1.15 mmol)
and a catalytic amount of boron trifluoride-ethyl ether complex
(BF3-OEt, 8.73 .mu.L, 0.069 mmol) were added dropwise at 0.degree.
C. After the reaction solution was stirred at room temperature for
another 30 minutes, it was diluted with dichloromethane and a
saturated aqueous sodium bicarbonate solution was added while
cooling on ice. After extraction with dichloromethane, the organic
layer was washed with saturated aqueous sodium chloride solution.
This organic layer was dried over sodium sulfate, the solvent was
distilled off under reduced pressure, and the resulting material
was further dried using a pump. The obtained residue was washed
twice with dichloromethane/hexane=1/1 (20 mL, 10 mL), and white
solids were removed by filtration. The obtained filtrate was
concentrated, the residue was purified by flash column
chromatography (purif pack (registered trademark) SIZE 20,
hexane/ethyl acetate, 0.1% diisopropylethyl amine (DIPEA)), and
(S)-methyl
2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-((2-phenylpro-
pan-2-yl)oxy)phenyl)propanoate (Compound 21, Fmoc-Tyr(3-F,Pis)-OMe;
210 mg, 0.379 mmol) was obtained in 83% yield. [0369] LCMS (ESI)
m/z=554.4 (M+H)+ [0370] Retention time: 1.09 minutes (analysis
condition SQDFA05)
Example 1-14
Synthesis of
(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-((2-pheny-
lpropan-2-yl)oxy)phenyl)propanoic acid (Compound 22,
Fmoc-Tyr(3-F,Pis)-OH)
##STR00046##
[0372] (S)-methyl
2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-((2-phenylpro-
pan-2-yl)oxy)phenyl)propanoate (Compound 21, Fmoc-Tyr(3-F,Pis)-OMe;
210 mg, 0.379 mmol) was dissolved in dichloroethane (DCE) (1.26
mL), trimethyltin(IV) hydroxide (Me3SnOH, 137 mg, 0.379 mmol) was
added, and this was stirred at 60.degree. C. for three hours. After
the reaction solution was concentrated using an evaporator, t-butyl
methyl ether (TBME, 2.0 mL) and 0.05 M aqueous phosphoric acid
solution (pH 2.1, 4.0 mL) were added, and this was stirred at
25.degree. C. for 15 minutes. After separating the organic layer,
the aqueous layer was extracted twice with t-butyl methyl ether
(TBME, 1 mL). The organic layer was dried over sodium sulfate, the
solvent was distilled off under reduced pressure, and the resulting
material was further dried using a pump. The obtained residue was
dissolved in a solution of 0.1% formic acid in acetonitrile,
stirred for 15 minutes, and then the obtained solution was purified
by reverse-phase chromatography (Wakosil 25C18, 30 g, 0.1% aqueous
formic acid solution/0.1% formic acid in acetonitrile) to obtain
(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino-3-(3-fluoro-4-((2-phenyl-
propan-2-yl)oxy)phenyl)propanoic acid (Compound 22,
Fmoc-Tyr(3-F,Pis)-OH; 190 mg, 0.352 mmol) in 93% yield. [0373] LCMS
(ESI) m/z=538.2 (M-H).sup.- [0374] Retention time: 1.00 minutes
(analysis condition SQDFA05)
Synthesis of Conjugates Formed Between Fmoc-Amino Acids and Resins
Used for Peptide Synthesis by a Peptide Synthesizer
[0375] Conjugates formed between Fmoc-amino acids and resins used
for peptide synthesis by a peptide synthesizer were synthesized as
follows.
Example 1-15
Synthesis of
(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-4-(piperidin-1-yl-
)butanoic acid-2-chlorotrityl resin (Compound 50,
Fmoc-Asp(O-Trt(2-Cl)-resin)-pip)
##STR00047##
[0377]
(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-4-(piperidi-
n-1-yl)butanoic acid-2-chlorotrityl resin (Compound 50,
Fmoc-Asp(O-Trt(2-Cl)-resin)-pip) was synthesized by a method
described in the literature (Document: WO 2013/100132 A1).
[0378] Herein, when a polymer or resin binds with a compound, the
polymer or resin moiety may be represented by ".smallcircle." (open
circle). Furthermore, to clarify the reaction point of the resin
moiety, the chemical structure of the reaction site may be shown by
connecting it to the open circle. For example, in the
above-mentioned structure (Fmoc-Asp(O-Trt(2-Cl)-resin)-pip
(Compound 50)), the 2-chlorotrityl group of the resin is linked to
the side-chain carboxylic acid of Asp through an ester bond.
Furthermore, pip means piperidine, and in the above-mentioned
structure, the C-terminal carboxylic acid group forms an amide bond
with piperidine.
Example 1-16
Synthesis of a Compound (Compound 52) in which the Side-Chain
Carboxylic Acid of Fmoc-Asp-piptBu (Compound 51) was Linked to a
Resin
Example 1-16-1
Synthesis of (S)-tert-butyl
3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(4-(tert-butyl)piperidin--
1-yl)-4-oxobutanoate (Compound 53, Fmoc-Asp(OtBu)-piptBu) (piptBu
Means 4-(tert-butyl)piperidine, and Here, it Shows that the
C-terminal Carboxylic Acid Group Forms an Amide Bond with
4-(tert-butyl)piperidine.)
##STR00048##
[0380]
(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4-
-oxobutanoic acid (10 g, 24.30 not), 4-(tert-butyl)piperidine
hydrochloride (4.10 g, 23.09 mmol), and 1-hydroxybenzotriazole
monohydrate (HOBt, 3.61 g) were dissolved in DMF (80 mL), then
1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride
(WSCI.HCl, 5.59 g) was added at 0.degree. C., and this was stirred
at 0.degree. C. for 30 minutes. Subsequently, 4-methylmorpholine
(2.54 mL) was added, and this was stirred at room temperature for
one hour. Hexane-ethyl acetate (1/1, v/v, 500 mL) was added to the
reaction solution, and the organic layer was washed twice with
saturated aqueous ammonium chloride solution, twice with saturated
aqueous sodium bicarbonate solution, and once with saturated
aqueous sodium chloride solution. The obtained organic layer was
dried over sodium sulfate, solids were removed by filtration, and
the solvent was distilled off under reduced pressure. The obtained
residue was purified by silica gel column chromatography (mobile
phase: hexane-ethyl acetate) to yield (S)-tert-butyl
3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(4-(tert-butyl)piperidin--
1-yl)-4-oxobutanoate (Compound 53, Fmoc-Asp(OtBu)-piptBu; 11.5 g,
21.51 mmol). [0381] LCMS (ES1) m/z=535.4 (M+H).sup.+ [0382]
Retention time: 1.17 minutes (analysis condition SQDAA05)
Example 1-16-2
Synthesis of
(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(4-(tert-butyl)piperi-
din-1-yl)-4-oxobutanoic acid (Compound 51, Fmoc-Asp-piptBu)
##STR00049##
[0384] Toluene was added to (S)-tert-butyl
3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(4-(tert-butyl)piperidin--
1-yl)-4-oxobutanoate (Compound 53, Fmoc-Asp(OtBu)-piptBu; 2.0 g,
3.74 mmol), and the included moisture was removed azeotropically by
distilling off the solvent is under reduced pressure. The obtained
residue was dissolved in dichloromethane (1.66 mL), and the
included amount of moisture was confirmed to be 110 ppm by the Karl
Fischer titration. Subsequently, the solution was stirred at
0.degree. C. for five minutes, trifluoroacetic acid (TFA, 1.66 mL)
was added dropwise at 0.degree. C., and this was stirred for five
minutes. The temperature of the reaction solution was brought back
to room temperature, and stirring was continued for four hours. The
mixture was cooled to 0.degree. C., and triethylamine (3.1 mL) was
added dropwise. The mixture was diluted with dichloromethane (30
mL), and this was washed with 5% aqueous sodium dihydrogenphosphate
solution (5% NaH2PO4aq, pH 4.4). The organic layer was dried over
sodium sulfate, solids were removed by filtration, and then the
solvent was distilled off under reduced pressure at 20.degree. C.
Since 19FNMR (DMSO-d6) measurement on the obtained residue
confirmed the presence of residual TFA, the residue was diluted
again in dichloromethane (30 mL), and this was washed with 5%
aqueous sodium dihydrogenphosphate solution (5% NaH2PO4aq, pH 4.4).
The organic layer was dried over sodium sulfate, the solids were
removed by filtration, and then the solvent was distilled off under
reduced pressure at 20.degree. C. to obtain
(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(4-(tert-butyl)piperi-
din-1-yl)-4-oxobutanoic acid (Compound 51, Fmoc-Asp-piptBu; 1.73
g). Residual TFA was confirmed to be below the detection limit by
19FNMR. [0385] LCMS (ESI) m/z=479.4 (M+H).sup.+ [0386] Retention
time: 1.00 minutes (analysis condition SQDAA05)
Example 1-16-3
Synthesis of
(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(4-(tert-butyl)piperi-
din-1-yl)-4-oxobutanoic acid-2-chlorotrityl resin (Compound 52,
Fmoc-Asp(O-Trt(2-Cl)-resin)-piptBu)
##STR00050##
[0388] 2-Chlorotritylchloride resin (1.60 mmol/g, 100-200 mesh, 1%
DVB, purchased from Watanabe Chemical Industries; 4.52 g, 7.23
mmol) and dehydrated dichloromethane (72 mL) were placed into a
reaction vessel equipped with a filter, and this was shaken at
25.degree. C. for ten minutes. After removing dichloromethane by
applying nitrogen pressure, a mixed solution produced by adding
dehydrated methanol (1.17 mL,) and diisopropylethylamine (DIPEA,
3.02 mL) to
(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(4-(tert-butyl)piperi-
din-1-yl)-4-oxobutanoic acid (Compound 51, Fmoc-Asp-piptBu; 1.73 g)
and dehydrated dichloromethane (72 mL) was added to the reaction
vessel, and this was shaken for 15 minutes. After removing the
reaction solution by applying nitrogen pressure, a mixed solution
produced by adding dehydrated methanol (9.0 mL) and
diisopropylethylamine (DIPEA, 3.02 mL) to dehydrated
dichloromethane (72 mL) was added to a reaction vessel, and this
was shaken for 90 minutes. After removing the reaction solution by
applying nitrogen pressure, dichloromethane was placed into the
vessel, and this was shaken for five minutes. The reaction solution
was removed by applying nitrogen pressure. This operation of
washing the resin with dichloromethane was repeated five times, and
the obtained resin was dried overnight under reduced pressure to
yield
(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(4-(tert-butyl)piperi-
din-1-yl)-4-oxobutanoic acid-2-chlorotrityl resin (Compound 52,
Fmoc-Asp(O-Trt(2-Cl)-resin)-piptBu, 5.23 g).
[0389] The obtained Fmoc-Asp(O-Trt(2-Cl)-resin)-piptBu (Compound
52, 16.5 mg) was placed into a reaction vessel, 20% piperidine/DMF
solution (1 mL) was added, and this was shaken at 25.degree. C. for
30 minutes. From the mixed reaction solution, 30 .mu.L was drawn
out and diluted using DMF (2.97 mL), its absorbance (301.2 nm) was
measured (Shimadzu, UV-1600PC (cell length: 1.0 cm) was used for
the measurement), and the loading rate of
Fmoc-Asp(O-Trt(2-Cl-resin)-piptBu (Compound 52) was calculated to
be 0.356 mmol/g.
[0390] Different lots synthesized similarly but having different
loading rates were also used for peptide synthesis.
Example 1-17
Synthesis of a Compound (Compound 55) in which Fmoc-Asp-MeOctyl
(Compound 54) was Linked to a Resin at its Side-Chain Carboxylic
Acid
Example 1-17-1
Synthesis of (S)-tert-butyl
3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(methyl(octyl)amino)-4-ox-
obutanoate (Compound 56, Fmoc-Asp(OtBu)-MeOctyl) (MeOctyl means
N-methyloctan-1-amine, and here, it Shows that the C-terminal
Carboxylic Acid Group Forms an Amide Bond with
N-methyloctan-1-amine)
##STR00051##
[0392]
(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino-4-(tert-butoxy)-4--
oxobutanoic acid (Fmoc-Asp(OtBu)-OH; 8.00 g, 19.44 mmol) and DNIF
(65 mL) were added to a 300-mL flask and stirred at room
temperature for five minutes. Next, 4-methylmorpholine (2.57 mL)
and O-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU; 8.87 g, 23.33 mmol) were added, and this
was stirred at 0.degree. C. for five minutes. Furthermore,
N-methyloctan-1-amine (3.35 mL, 18.47 mmol) was added dropwise over
a period of two minutes, and the obtained reaction solution was
stirred at 0.degree. C. for 30 minutes. To this reaction solution,
hexane-ethyl acetate (1/1, v/v, 400 mL) was added, and this was
washed with water (400 mL), saturated aqueous ammonium chloride
solution (400 mL), 50% aqueous sodium bicarbonate solution (400
mL), water (400 mL.times.2), and then saturated aqueous sodium
chloride solution (400 mL). The obtained organic layer was dried
over sodium sulfate, solids were removed, and the solvent was
distilled off under reduced pressure. The obtained residue was
purified by silica gel column chromatography (hexane-ethyl acetate)
to yield (S)-tert-butyl
3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(methyl(octyl)amino)-4-ox-
obutanoate (Compound 56, Fmoc-Asp(OtBu)-MeOctyl; 10.2 g, 19.00
mmol). [0393] LCMS (ESI) m/z=537.5 (M+H).sup.30 [0394] Retention
time: 0.84 minutes (analysis condition SQDFA50)
Example 1-17-2
Synthesis of
(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(methyl(octyl)amino)--
4-oxobutanoic acid (Compound 54, Fmoc-Asp-MeOctyl)
##STR00052##
[0396] Toluene was added to (S)-tert-butyl
3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(methyl(octyl)amino)-4-ox-
obutanoate (Compound 56, Fmoc-Asp(OtBu)-MeOctyl; 8.1 g, 15.09
mmol), and the included moisture was removed azeotropically by
distilling off the solvent under reduced pressure. The obtained
residue was dissolved in dichloromethane (dehydrated, 6.7 and the
included amount of moisture was confirmed to be 380 ppm by the Karl
Fischer titration. Subsequently, the solution was stirred at
0.degree. C. for five minutes, trifluoroacetic acid (TFA, 6.7 mL)
was added dropwise at 0.degree. C. over a period of five minutes,
and then this was stirred for five minutes. The temperature of the
reaction solution was brought back to room temperature, and
stirring was continued for four hours. The mixture was cooled to
0.degree. C., and triethylamine (12.62 mL) was added dropwise. The
mixture was diluted with dichloromethane (100 mL), and this was
washed with 5% aqueous sodium dihydrogenphosphate solution (5%
NaH2PO4aq). The organic layer was dried over sodium sulfate, solids
were removed by filtration, and then the solvent was distilled off
under recued pressure at 20.degree. C. Since 19FNMR (DMSO-d6)
measurement on the obtained residue confirmed the presence of
residual TFA, the residue was dissolved again in dichloromethane,
and this was washed with 5% aqueous sodium dihydrogenphosphate
solution (5% NaH2PO4aq). The organic layer was dried over sodium
sulfate, the solids were removed by filtration, and then the
solvent was distilled off under reduced pressure at 20.degree. C.
to obtain
(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(methyl(octyl)-
amino)-4-oxobutanoic acid (Compound 54, Fmoc-Asp-MeOctyl; 6.61 g,
13.75 mmol). Residual TFA was confirmed to be below the detection
limit by 19FNMR. [0397] LCMS (ESI) m/z=481.4 (M+H).sup.+ [0398]
Retention time: 0.65 minutes (analysis condition SQDAA50)
Example 1-17-3
Synthesis of
(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(methyl(octyl)amino)--
4-oxobutanoic acid-2-chlorotrityl resin (Compound 55,
Fmoc-Asp(O-Trt(2-Cl)-resin)-MeOctyl)
##STR00053##
[0400] 2-Chlorotritylchloride resin (1.60 mmol/g, 100-200 mesh, 1%
DVB, purchased from Watanabe Chemical Industries; 16.3 g, 26.1
mmol) and dehydrated dichloromethane (261 mL) were placed into a
reaction vessel equipped with a filter, and this was shaken at
25.degree. C. for ten minutes. After removing dichloromethane by
applying nitrogen pressure, a mixed solution produced by adding
dehydrated methanol (4.23 mL) and diisopropylethylamine (DIPEA,
10.9 mL) to
(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(methyl(octyl)amino)--
4-oxobutanoic acid (Compound 54, Fmoc-Asp-MeOctyl, 6.28 g, 13.07
mmol) and dehydrated dichloromethane (261 mL) was added to the
reaction vessel, and this was shaken for 15 minutes. After removing
the reaction solution by applying nitrogen pressure, a mixed
solution produced by adding dehydrated methanol (32.4 mL) and
diisopropylethylamine (DIPEA, 10.9 mL) to dehydrated
dichloromethane (261 mL) was added to the reaction vessel, and this
was shaken for 90 minutes. After removing the reaction solution by
applying nitrogen pressure, dichloromethane (261 mL) was placed
into the vessel, and this was shaken for five minutes. The reaction
solution was removed by applying nitrogen pressure. This operation
of washing the resin with dichloromethane was repeated twice, and
the obtained resin was dried overnight under reduced pressure to
yield
(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(methyl(octyl)amino)--
4-oxobutanoic acid-2-chlorotrityl resin (Compound 55,
Fmoc-Asp(O-Trt(2-Cl)-resin)-MeOctyl; 18.2 g). [0401] Loading rate:
0.366 mmol/g
[0402] Different lots synthesized similarly but having different
loading rates were also used for peptide synthesis.
Example 1-18
Synthesis of a Compound (Compound 58) in which Fmoc-Asp-Pro-OPis
(Compound 57) was Linked to a Resin at its Side-Chain Carboxylic
Acid
Example 1-18-1
Synthesis of (S)-2-phenylpropan-2-yl
1-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(allyloxy)-4-oxobu-
tanoyl)pyrrolidine-2-carboxylate (Compound 59,
Fmoc-Asp(OAll)-Pro-OPis)
##STR00054##
[0404] A solution of 2-(2-phenylpropan-2-yl)
(S)-1((9H-fluoren-9-yl)methyl) pyrrolidine-1,2-dicarboxylate
(Compound 14, Fmoc-Pro-OPis; 20.0 g, 43.9 mmol) prepared by the
already described method in dehydrated DMF (40 mL) was cooled to
20.degree. C. using a water bath.
1,8-Diazabicyclo[5.4.0]-7-undecene (DBU; 6.57 mL, 43.9 mmol) was
added to this solution dropwise over a period of seven minutes, and
this was stirred at room temperature for five minutes.
Subsequently, the reaction solution was cooled to 0.degree. C.,
pyridine hydrochloride (5.07 g, 43.9 mmol) was added, and this was
stirred at 0.degree. C. for ten minutes. Thereafter, a mixture of
(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(allyloxy)-4-oxobutan-
oic acid (Fmoc-Asp(OAll)-OH; 17.35 g, 43.9 mmol),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(WSCI.HCl; 11.8 g, 61.4 mmol), and 1-hydroxy-7-azabenzotriazol
(HOAt; 7.17 g, 52.7 mmol) was added, and additionally,
diisopropylethylamine (DIPEA, 7.6 mL, 43.9 mmol) was added dropwise
at 0.degree. C. over a period of seven minutes. The reaction
solution was stirred at room temperature for 20 minutes. To the
obtained reaction solution, hexane (50 mL), diethyl ether (50 mL),
saturated aqueous sodium bicarbonate solution (10 mL), and
saturated aqueous sodium chloride solution (50 mL) were added, and
the aqueous layer was extracted twice using diethyl ether. All of
the obtained organic layers were combined, and washed three times
with saturated aqueous sodium chloride solution (50 mL), and then
this was dried over sodium sulfate. The solvent was removed under
reduced pressure, and the obtained residue was purified by silica
gel column chromatography (mobile phase: hexane-ethyl acetate) to
yield (S)-2-phenylpropan-2-yl
1-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(allyloxy)-4-oxobu-
tanoyl)pyrrolidine-2-carboxylate (Compound 59,
Fmoc-Asp(OAll)-Pro-OPis; 24.5 g, 40.1 mmol), [0405] LCMS (ESI)
m/z=611.4 (M+H).sup.+ [0406] Retention time: 1.03 minutes (analysis
condition SQDFA05)
Example 1-18-2
Synthesis of
(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-4-((S)-2-(((2-phe-
nylpropan-2-yl)oxy)carbonyl)pyrrolidin-1-yl)butanoic acid (Compound
57, Fmoc-Asp-Pro-OPis)
##STR00055##
[0408] (S)-2-phenylpropan-2-yl
1-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(allyloxy)-4-oxobu-
tanoyl)pyrrolidine-2-carboxylate (Compound 59,
Fmoc-Asp(OAll)-Pro-OPis; 24.16 g, 39.6 mmol) and
tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4; 0.114 g, 0.099
mmol) were placed in a 300-mL two-necked flask, and the interior of
the flask was nitrogen substituted. Subsequently, dichloromethane
(40 mL) was added, this was stirred at room temperature, and then
this was cooled to 14.degree. C. in a water bath. Phenylsilane
(3.30 mL, 26.7 mmol) was added dropwise over a period of five
minutes, and the reaction solution was stirred under nitrogen
atmosphere at 14.degree. C. to 17.degree. C. for 35 minutes. Next,
SH silica (manufactured by Fuji Silysia; 5 g) and methanol (32.1
mL) were added, and then Kieselgel 60 (15 g) was added. Methanol
(30 mL), SH silica (manufactured by Fuji Silysia; 5 g) and
Kieselgel 60 (25 g) were further added, and the mixture was stirred
at 17.degree. C. to 24.degree. C. until the liquid phase became
colorless. The obtained mixture was filtered through Celite and
washed with dichloromethane-methanol (10/1, v/v), and the solvent
was distilled off under reduced pressure to yield
(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-4-((S)-2-(((2-phe-
nylpropan-2-yl)oxy)carbonyl)pyrrolidin-1-yl)butanoic acid (Compound
57, Fmoc-Asp-Pro-OPis) as a crude product (25.38 g). The obtained
crude product was used without purification in the subsequent
process of making the resins support the compound. [0409] LCMS
(ESI) m/z=571.3 (M+H).sup.+ [0410] Retention time: 0.88 minutes
(analysis condition SQDFA05)
Example 1-18-3
Synthesis of
(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-4-((S)-2-(((2-phe-
nylpropan-2-yl)oxy)carbonyl)pyrrolidine-1-yl)butanoic
acid-2-chlorotrityl resin (Compound 58,
Fmoc-Asp(O-Trt(2-Cl)-resin)-Pro-OPis)
##STR00056##
[0412] 2-Chlorotritylchloride resin (1.60 mmol/g, 100-200 mesh, 1%
DVB, purchased from Watanabe Chemical Industries; 47.8 g, 76.48
mmol) and dehydrated dichloromethane (150 mL) were placed into a
reaction vessel equipped with a filter, and this was shaken at
2.5.degree. C.. for 35 minutes. After removing dichloromethane by
applying nitrogen pressure, a mixed solution produced by adding
dehydrated methanol (3.11 mL) and diisopropylethylamine (DIPEA,
32.1 mL) to a prepared solution of
(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-4-((S)-2-(((2-phe-
nylpropan-2-yl)oxy)carbonyl)pyrrolidin-1-yl)butanoic acid (Compound
57, Fmoc-Asp-Pro-OPis; 21.94 g, 38.4 mmol) in dehydrated
dichloromethane (115 mL) was added to a reaction vessel, and this
was shaken for 45 minutes. After removing the reaction solution by
applying nitrogen pressure, a mixed solution produced by adding
dehydrated methanol (55 mL) and diisopropylethylamine (DIPEA, 25
mL) to dehydrated dichloromethane (100 mL) was added to the
reaction vessel, and this was shaken for 90 minutes. After removing
the reaction solution by applying nitrogen pressure,
dichloromethane (100 mL) was placed into the vessel, and this was
shaken for five minutes. The reaction solution was removed by
applying nitrogen pressure. This operation of washing the resin
with dichloromethane (100 mL) was repeated four times, and the
obtained resin was dried for 15.5 hours under reduced pressure to
yield
(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-4-((S)-2-(((2-phe-
nylpropan-2-yl)oxy)-carbonyl)pyrrolidine-1-yl)butanoic
acid-2-chlorotrityl resin (Compound 58,
Fmoc-Asp(O-Trt(2-Cl)-resin)-Pro-OPis 61.55 g).
[0413] The obtained Fmoc-Asp(O-Trt(2-Cl)-resin)-Pro-OPis (Compound
58, 12.3 mg) was placed into a reaction vessel, DMF (0.2 mL) and
piperidine (0.2 mL) were added, and this was shaken at 25.degree.
C. for 30 minutes. DMF (1.6 mL) was added to the reaction vessel,
then 0.4 mL of the reaction mixture solution was drawn out, this
was diluted with DMF (9.6 mL). and its absorbance (301.2 nm) was
measured (Shimadzu. UV-1600PC (cell length: 1.0 cm) was used for
the measurement). From the following calculation formula, the
loading rate of Fmoc-Asp(O-Trt(2-Cl)-resin)-Pro-OPis (Compound 58)
was calculated to be 0.3736 mmol/g.
[0414] (Absorption (301.2 nm).times.1000.times.50)/(resin weight
(mg).times.7800)=(0.717.times.1000.times.50)/(12.3.times.7800)=0.3736
mmol/g
[0415] Different lots synthesized similarly but having different
loading rates were also used for peptide synthesis.
Example 2
Chemical Synthesis of Peptides by a Peptide Synthesizer (Steps A to
C)
[0416] Unless otherwise stated particularly, peptide synthesis by
the above-mentioned basic synthetic route was performed by the
following methods.
Example 2-1
Solid-Phase Synthesis of Peptides by an Automated Synthesizer (Step
A)
[0417] Peptide synthesis by the Fmoc method was performed using a
peptide synthesizer (Multipep RS; manufactured by Intavis).
Specific procedures for the operation were performed according to
the instructions attached to the synthesizer.
[0418] A solution of 2-chlorotritylresin (100 mg per column) linked
to the side-chain carboxylic acid portion of an aspartic acid in
which its N terminus is protected with Fmoc, various Fmoc-amino
acids (0.6 mol/L, 0.5 mol/L in the case of Fmoc-MeHis(Trt)-OH), and
1-hydroxy-7-azabenzotriazole (HOAt) or oxyma (0.375 mol/L) in NMP,
and a solution of diisopropylcarbodiimide (DIC) in
N,N-dimethylformaldehyde (DMF) (10% v/v) were placed into the
synthesizer. Furthermore, when the Fmoc-amino acid used was
Fmoc-Ser(THP)-OH (Compound 1), Fmoc-Thr(THP)-OH (Compound 2), or
Fmoc-MeSer(THP)-OH (Compound 6), such an Fmoc-amino acid was made
to coexist with oxyma in the NMP solution, and it was placed into
the synthesizer after further adding Molecular Sieves 4A 1/8 (Wako
Pure Chemical Industries) or Molecular Sieves 4A 1/16 (Wako Pure
Chemical Industries).
[0419] A solution of diazabicycloundecene (DBU) was used as an in
DMF (2% v/v) was used as an Fmoc deprotection solution. After
washing the resin with DMF, the Fmoc groups were deprotected, and
then condensation reaction with new Fmoc amino acid was performed.
This was defined as a single cycle, and by repeating this cycle
multiple times, the peptides were elongated on the surface of the
resin.
Example 2-2
Cleavage of the Elongated Peptides From the Resin (Step B)
[0420] The Fmoc groups of the N terminus of the peptides elongated
by the above-mentioned method were removed on the peptide
synthesizer, and then the resin was washed with DMF. This was
followed by swelling the resin again in DCM, and then after adding
TFE/DCM (1/1, v/v, 2 mL) to the resin, and this was shaken at room
temperature for two hours. Subsequently, the solution in the tube
was filtered through a column for synthesis to remove the resin,
and the remaining resin was further washed twice with TFE/DCM (1/1,
v/v, 1 mL). All the cleavage solutions obtained were mixed, and
this was concentrated under reduced pressure.
Example 2-3
Cyclization of the Cleaved Peptides (Step C)
[0421] The residue resulting from cleavage followed by
concentration under reduced pressure was dissolved in DMF/DCM (1/1,
v/v, 8 mL). A 0.5 M
O-(7-aza-1H-benzotriazol-1-yl)-N,N,N,N-tetramethyluronium
hexafluorophosphate (HATU)/DMF solution (volume corresponding to
1.5 equivalents with respect to the number of moles on the resin
used (loading rate (mmol/g) multiplied by the amount of resin used
(normally 0.10 g)) and DIPEA (1.8 equivalents with respect to the
number of moles on the resin used) were added, and this was shaken
at room temperature for two hours. Thereafter, the solvent was
distilled off under reduced pressure. Generation of the desired
cyclic peptides was confirmed by LCMS analyses.
[0422] Peptides Pep1 to Pep7 to be used for examination of the
later-described deprotection reactions were synthesized by the
above-mentioned methods. The sequences, structures, and LCMS data
of Pep1 to Pep7 are shown in Tables 2-1, 2-2, and 2-3,
respectively. Examinations of deprotection conditions (degrees of
hydrolysis and N- to O-acyl shift problems observed during
deprotection, or peptides to be coexisted in the solution which are
to be examined) carried out later were evaluated using residues
containing the cyclic peptides obtained in this process.
TABLE-US-00002 TABLE 2-1 MW EM (Cyclized) (Cyclized) 11 10 9 8 7
Pep-1 Compound 101 1490.2 1478.75 Ala Trp Nle Trp D-Tyr(tBu) Pep-2
Compound 102 1852.4 1581.11 MePhe MePhe Leu MeLeu Thr(THP) Pep-3
Compound 103 2018.8 2016.97 g-EtAbu MeSer(DMT) Hyp(Et) Ile
MePhe(3-Cl) Pep-4 Compound 104 1344.0 1342.66 Ala Trp Nle Trp MeAla
Pep-5 Compound 105 1574.3 1572.74 Ala Trp Nle Trp Ser(Trt) Pep-6
Compound 106 1775.1 1773.56 g-EtAbu Hyp(Et) MeAla(4-Th ) MeAla
MeSer(DMT) Pep-7 Compound 107 1734.0 1732.37 Ala Phe(4-CF3) Trp Trp
MeLeu Used Resin 6 5 4 3 2 1 H-1 Resin Loading Amount Pep-1 MeGly
MeAla MePhe(3-Cl) MeGly nPrGly Asp pip Compound 50 0.342 mmol/g
Pep-2 MeLeu MeLeu His(Trt) MePhe MeLeu Asp pip Compound 50 0.368
mmol/g Pep-3 Ser(Trt) Trp Trp Pro MeGly Asp pip Compound 50 0.318
mmol/g Pep-4 MeGly MeAla MePhe(3-Cl) MeGly Pro Asp pip Compound 50
0.342 mmol/g Pep-5 Gly MeAla MePhe(3-Cl) MeGly Pro Asp pip Compound
50 0.342 mmol/g Pep-6 Hyp(Et) Trp Trp Pro MeGly Asp pip Compound 50
0.318 mmol/g Pep-7 MeGly MeGly Pro Hyp(Et) Ser(Trt) Asp piptBU
Compound 52 0.358 mmol/g indicates data missing or illegible when
filed
TABLE-US-00003 TABLE 2-2 Pep-1 Compound 101 ##STR00057## Pep-2
Compound 102 ##STR00058## Pep-3 Compound 103 ##STR00059## Pep-4
Compound 104 ##STR00060## Pep-5 Compound 105 ##STR00061## Pep-6
Compound 106 ##STR00062## Pep-7 Compound 107 ##STR00063##
TABLE-US-00004 TABLE 2-3 LCMS Retention Condition Time (min) LCMS
(ESI) m/z Pep-1 Compound 101 SQDFA05 0.93 1480.1 (M + H)+ Pep-2
Compound 102 SQDFA05 1.00 1852.3 (M + H)+ Pep-3 Compound 103
SQDFA05 0.78 (30%), 1474.1, 1.06 (70%) 1716.2 (M + H)+ Pep-4
Compound 104 SQDFA05 0.78 1343.9 (M + H)+ Pep-5 Compound 105
SQDFA05 1.01 1574.2 (M + H)+ Pep-6 Compound 106 SQDFA05 0.98 1473.1
(M + H)+ Pep-7 Compound 107 SQDFA05 1.10 1734.2 (M + H)+
Example 3
Deprotection of Protecting Groups on Peptide Side-Chain Functional
Groups Using Weakly Acidic Solutions Produced from Weak Acids
(having Aqueous pKa of 0 to 9) and Solvents (Solvents which have
Positive Y.sub.OTs Values, are Weakly Acidic (Aqueous pKa of 5 to
14) and have Low Nucleophilicity) (Step D)
Example 3-1
Possibility of Deprotection of Protecting Groups on Fmoc-Amino Acid
Side-Chain Functional Groups Using the Above-Mentioned Weakly
Acidic Solution
[0423] Whether the protecting groups of side-chain functional
groups of Fmoc-amino acids used in peptide synthesis can be
deprotected under conditions of weaker acidity than TFA, more
specifically, in a solution produced by dissolving a weak acid
having an aqueous pKa of 0 to 9 in a solvent that has a positive
YOTs value, is weakly acidic (aqueous pKa of 5 to 14), and has low
nucleophilicity, was examined.
[0424] Specifically, tetramethylammonium hydrogen sulfate (pKa 2.0)
was used as the weak acid and HFIP (having a YOTs value of 3.82
(value based on the literature: Prog. Phys. Org. Chem. 1990, 17,
121-158), and a pKa of 9.30) was used as the solvent. More
specifically, either 0.1 M tetramethylammonium hydrogen
sulfate/HFIP solution (2% TIPS) or 0.05 M tetramethylammonium
hydrogen sulfate/HFIP solution (2% TIPS) used.
Example 3-1-1
Preparation of 0.1 M tetramethylammonium hydrogen sulfate/HFIP
solution (2% TIPS)
[0425] The 0.1 M tetramethylammonium hydrogen sulfate/HFIP solution
TIPS) was prepared by drawing 4 mL of a solution produced by mixing
HFIP (11.66 mL), TIPS (0.24 mL), and DCE (0.10 mL), and dissolving
68.5 mg of tetramethylammonium hydrogen sulfate in this
solution.
Example 3-1-2
Preparation of 0.05M tetramethylammonium hydrogen sulfate/HFIP
Solution (2% TIPS)
[0426] The 0.05 M tetramethylammonium hydrogen sulfate/HFIP
solution (2% TIPS) was prepared by drawing 4 mL of a solution
produced by mixing HFIP (11.66 mL), TIPS (0.24 mL), and DCE (0.10
mL), and dissolving 34.3 mg of tetramethylammonium hydrogen sulfate
in this solution.
[0427] Fmoc-amino acids having side-chain protecting groups or
peptides which comprise amino acid residues having side-chain
protecting groups were deprotected by method A or method B
described below.
Example 3-1-3
Method A
[0428] To a mixture of an Fmoc-amino acid (4.0 .mu.mol) and a
peptide (any one of the already synthesized cyclic peptides Pep 1
to Pep 6 (residue after cyclization); maximum concentration of 3.66
.mu.mol) having protecting groups on their side chains, 0.1 M
tetramethylammonium hydrogen sulfate/HFIP solution (2% TIPS) (0.20
mL) or 0.05 M tetramethylammonium hydrogen sulfate/HFIP solution
(2% TIPS) (0.40 mL)was added and shaken for three minutes, then
this was left to stand at 25.degree. C. After a given amount of
time, LCMS (FA05) analyses were carried out. Progress of the
deprotection was calculated from the UV area ratio of the
deprotected peptide to the protected peptide.
Example 3-1-4
Method B
[0429] To a peptide (any one of the already synthesized cyclic
peptides Pep 1 to Pep 6 (residue after cyclization); maximum
concentration of 3.66 .mu.mol) comprising amino acid residues
having protecting groups on their side chains, 0.1 M
tetramethylammonium hydrogen sulfate/HFIP (2% TIPS) (0.20 mL) or
0.05 M tetramethylammonium hydrogen sulfate/HFIP (2% TIPS) (0.40
mL) was added and shaken for three minutes. This was left to stand
at 25.degree. C., and after a given amount of time. LCMS (FA05)
analyses were carried out. Progress of the deprotection was
calculated from the UV area ratio of the deprotected peptide to the
protected peptide.
[0430] The peptides prepared as follows were used in method A and
method B: elongation was performed and elongated peptides were
cleaved off from the resin by the already described method and
additionally subjected to a cyclization reaction by the already
described method; then peptides were concentrated under reduced
pressure, and the obtained residues were dissolved in
dichloromethane, and this was aliquoted into 10 test tubes; and
then concentrated again by removing the solvent under reduced
pressure.
[0431] The results of evaluation are as shown below in Table 3.
TABLE-US-00005 TABLE 3 Protected Time Deprotection run Amino Add
method Coexisted Peptide Condition (h) (%) 1 D-Tyr (tBu) B Pep 1
(Compound 101) 0.1M 24 >99% 2 B Pep 1 (Compound 101) 0.05M 24
81% 3 Tyr (3-F, tBu) A Pep 2 (Compound 102) 0.1M 22 65% 4 A Pep 2
(Compound 102) 0.05M 19 19% 5 MeHis (Trt) A Pep 2 (Compound 102)
0.05M 4 >99% 6 Thr (THP) B Pep 2 (Compound 102) 0.05M 2 >99%
7 MeSer (DMT) B Pep 3 (Compound 103) 0.05M 4 >99% 8 MeSer (THP)
A Pep 4 (Compound 104) 0.05M 1 >99% 9 Ser (Trt) B Pep 5
(Compound 105) 0.05M 4 >99% 10 Ser (THP) A Pep 3 (Compound 103)
0.05M 1 >99% 11 Pro-OPis A Pep 2 (Compound 102) 0.05M 2 >99%
12 D-Tyr (Clt) A Pep 5 (Compound 105) 0.05M 1 >99% 13 D-Tyr
(THP) A Pep 6 (Compound 106) 0.05M 1.25 >99% 14 D-Tyr (Pis) A
Pep 6 (Compound 106) 0.05M 1.25 >99% 15 Tyr (3-F, Pis) A Pep 3
(Compound 103) 0.05M 1 >99%
[0432] Results of LCMS analyses taken on Fmoc-amino acids after
deprotection are as follows: [0433] Fmoc-Tyr(3-F)-OH (deprotection
product of run 3, run 4, and run 15) [0434] LCMS (ESI) m/z=422.3
(M+H).sup.+ [0435] Retention time: 0.73 minutes (analysis condition
SQDFA05) [0436] Fmoc-MeHis-OH (deprotection product of run 5)
[0437] LCMS (ESI) m/z=392.3 (M+H).sup.+ [0438] Retention time: 0.47
minutes (analysis condition SQDFA05) [0439] Fmoc-MeSer-OH
(deprotection product of run 8) [0440] LCMS (ESI) m/z=342.3
(M+H).sup.+ [0441] Retention time: 0.67 minutes (analysis condition
SQDFA05) [0442] Fmoc-Ser-OH (deprotection product of run 10) [0443]
LCMS (ESI) m/z=328.2 (M+H).sup.+ [0444] Retention time: 0.64
minutes (analysis condition SQDFA05) [0445] Fmoc-Pro-OH
(deprotection product of run 11) [0446] LCMS (ESI) m/z=338.3
(M+H).sup.+ [0447] Retention time: 0.75 minutes (analysis condition
SQDFA05) [0448] Fmoc-D-Tyr-OH (deprotection product of run 12 to
run 14) [0449] LCMS (ESI) m/z=404.3 (M+H).sup.+ [0450] Retention
time: 0.72 minutes (analysis condition SQDFA05)
##STR00064##
[0451] Deprotection product of Pep 1 (Compound 101) (deprotection
product of run 1 and run 2; Compound 131) [0452] LCMS (ESI)
m/z=1424.0 (M+H).sup.+ [0453] Retention time: 0.79 minutes
(analysis condition SQDFA05)
##STR00065##
[0454] Deprotection product of Pep 2 (Compound 102) (deprotection
product of run 6; Compound 132) [0455] LCMS (ESI) m/z=1526.3
(M+H).sup.+ [0456] Retention time: 0.89 minutes (analysis condition
SQDFA05)
##STR00066##
[0457] Deprotection product of Pep 3 (Compound 103) (deprotection
product of run 7; Compound 133) [0458] LCMS (ESI) m/z=1474.1
(M+H).sup.+ [0459] Retention time: 0.78 minutes (analysis condition
SQDFA05)
##STR00067##
[0460] Deprotection product of Pep 5 (Compound 105) (deprotection
product of run 9; Compound 135) [0461] LCMS (ESI) m/z=1331.7
(M+H).sup.+ [0462] Retention time: 0.74 minutes (analysis condition
SQDFA05)
[0463] From these results, these protecting groups were confirmed
to undergo deprotection under a condition of 0.1 M
tetramethylammonium hydrogen sulfate/HFIP (2% TIPS) or 0.05 M
tetramethylammonium hydrogen sulfate/HFIP (2% TIPS).
[0464] Protecting groups on the side chains are not affected by
peptide cleavage conditions that use a TFE-DCM (1/1, v/v) solution
or a TFE-DCM (1/1, v/v)/DIPEA (addition of 1.8 equivalents to the
theoretical number of moles yielded by multiplying the loading rate
of the resin used by the amount of resin used) solution. Therefore,
in the intramolecular cyclization at the peptide main-chain N
terminus and the Asp side-chain carboxylic acid portion after the
cleavage step, the amino acid side-chain functional group is
maintained in the protected form. As a result, undesired
cyclization reactions in which the amino acid side-chain functional
group functions as the nucleophilic species can be suppressed.
3-2
Possibility of Suppressing Hydrolysis and N- to O-acyl Shift
Product Generation which Take Place when Protecting Groups of
Fmoc-Amino Acid Side-Chain Functional Groups are Deprotected Using
the Above-Mentioned Weakly Acidic Solution
Example 3-2-1
Deprotection of the Cyclic Compound (Compound 101, Pep 1), in which
an Amide Bond was Formed Between the N-Terminal Amino Group of
H-Ala-Trp-Nle-Trp-D-Tyr(tBu
-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip and the Side-Chain
Carboxylic Acid of Asp, by Using 0.1 M tetramethylammonium hydrogen
sulfate/HFIP Solution (2% TIPS) as the Deprotection Condition
[0465] Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (Compound 50, resin loading
rate: 0.342 mmol/g, 100 mg) was used as the resin, and cyclic
compound (Compound 101) in which an amide bond is formed between
the N-terminal amino group of
H-Ala-Trp-Nle-Trp-D-Tyr(tBu)-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip
and the side-chain carboxylic acid of Asp was synthesized by the
already described method. After cyclization, a residue formed by
concentration under reduced pressure was dissolved in
dichloromethane, then this was aliquoted into 10 test tubes, and
then they were concentrated again by removing the solvent under
reduced pressure.
[0466] To one of the 10 test tubes aliquoted, 0.20 mL of 0.1 M
tetramethylammonium hydrogen sulfate/HFIP solution (2% TIPS) (68.5
mg of tetramethylammonium hydrogen sulfate was dissolved in 4 mL of
solution drawn out from a solution produced by mixing HFIP: 11.66
mL, TIPS: 0.24 mL, and DCE: 0.10 mL) was added. The test tube was
capped using a rubber septum, shaken for three minutes, and then
left to stand at 25.degree. C. for 24 hours. When the reaction was
confirmed by LCMS (FA05), completion of side-chain deprotection
(deprotection of the tBu group of D-Tyr(tBu)) could be confirmed.
Here, the ratio among the deprotected desired peptide (Compound
131), the solvolysis product (compound showing a mass spectrum in
which any of the amide bonds of the peptide has undergone
solvolysis by the solvent HFIP), and the hydrolysate (compound
showing a mass spectrum in which any of the amide bonds of the
peptide has undergone solvolysis by water) was 72:10:18 (FIG. 2).
In the Examples, "TM+H2O" represents a compound in which any one of
the amide bonds of the target molecule has undergone hydrolysis,
and "TM+HFIP" represents a compound in which any one of the amide
bonds of the target molecule has undergone solvolysis by HFIP.
[0467] The data of FIG. 2 are shown below: [0468] Desired peptide
(Compound 131) [0469] LCMS (ESI) m/z=1424.0 (M+H)+ [0470] Retention
time: 0.79 minutes (analysis condition SQDFA05) [0471] Hydrolysate
(TM+H2O) [0472] LCMS (ESI) m/z=1442.0 (M+H)+ [0473] Retention time:
0.61 minutes (analysis condition SQDFA05) [0474] Product of
solvolysis by HFIP (TM+HFIP) [0475] LCMS (ESI) m/z=1592.0 (M+H)+
[0476] Retention time: 0.69 minutes, 0.71 minutes (analysis
condition SQDFA05)
Example 3-2-2
Deprotection of the Cyclic Compound (Compound 101, Pep1) in which
an Amide Bond was Formed Between the N-Terminal Amino Group of
H-Ala-Trp-Nle-Trp-D-Tyr(tBu)-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip
and the Side-Chain Carboxylic Acid of Asp, by Using 0.05 M
tetramethylammonium hydrogen sulfate/HFIP Solution (2% TIPS) as the
Deprotection Condition
[0477] After synthesizing Compound 101 (Pep1), 0.40 mL of 0.05 M
tetramethylammonium hydrogen sulfate/HFIP solution (2% TIPS) (34.3
mg of tetramethylammonium hydrogen sulfate was dissolved in 4 mL of
solution drawn out from a solution produced by mixing HFIP: 11.66
mL, TIPS: 0.24 mL, and DCE: 0.10 mL) was added to another one of
the 10 test tubes aliquoted in the above-mentioned operation. The
test tube was capped using a rubber septum, shaken for three
minutes, and then left to stand at 25.degree. C. for 24 hours, and
then the reaction was checked by LCMS (FA05). As a result,
side-chain deprotection (deprotection of the tBu group of
D-Tyr(tBu)) had proceeded 81%, and at this time, the ratio among
the deprotected desired peptide (Compound 131), the solvolysis
product (compound showing a mass in which any of the amide bonds of
the peptide has undergone solvolysis by the solvent HFIP), and the
hydrolysate (compound showing a mass in which any of the amide
bonds of the peptide has undergone hydrolysis by water) was 93:3:4
(FIG. 3). "TM+H.sub.2O" represents a compound in which any one of
the amide bonds of the target molecule has undergone hydrolysis.
Similarly, "TM+HFIP" represents a compound in which any one of the
amide bonds of the target molecule has undergone solvolysis by
HFIP.
[0478] The data of FIG. 3 are shown below: [0479] Desired peptide
(Compound 131) [0480] LCMS (ESI) m/z=1424.1 (M+H)+ [0481] Retention
time: 0.79 minutes (analysis condition SQDFA05) [0482] Hydrolysate
[0483] LCMS (ESI) m/z=1442.0 (M+H)+ [0484] Retention time: 0.61
minutes (analysis condition SQDFA05) [0485] Product of solvolysis
by HFIP [0486] LCMS (ESI) m/z=1592.0 (M+H)+ [0487] Retention time:
0.69 minutes, 0.71 minutes (analysis condition SQDFA05)
[0488] As described in the comparative example below, when the
cyclic compound (Compound 101, Pep1) in which an amide bond was
formed between the N-terminal amino group of
H-Ala-Trp-Nle-Trp-D-Tyr(tBu)-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip
and the side-chain carboxylic acid of Asp was deprotected under
conditions of 5% TFA/DCE, deprotection for 2.5 hours at 25.degree.
C. resulted in 87% progress of the hydrolysis.
[0489] On the other hand, by using 0.1 M or 0.05 M
tetramethylammonium hydrogen sulfate/HFIP solution (2% TIPS)
instead of 5% TFA on the same peptide sequence, production of
hydrolysates (and solvolysis products) could be reduced
significantly. Furthermore, this result suggests the possibility
that if a condition yielding weaker acidity than TFA can be
satisfied, the concentration of the weak acid can be adjusted as
one chooses.
Example 3-2-3
Deprotection of the Cyclic Compound (Compound 103, Pep3) in which
an Amide Bond is Formed Between the N-Terminal Amino Group of
H-g-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3-Cl)-Ser(Trt)-Trp-Trp-Pro-MeGly-A-
sp-pip and the Side-Chain Carboxylic Acid of Asp, by Using 0.05 M
tetramethylammonium hydrogen sulfate/HFIP Solution (2% TIPS) as the
Deprotection Condition
[0490] Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (Compound 50, loading: 0.316
mmol/g, 100 mg) was used as the resin, and the cyclic compound
(Compound 103, Pep3) in which an amide bond was formed between the
N-terminal amino group of
H-g-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3-Cl)-Ser(Trt)-Trp-Trp-Pr-
o-MeGly-Asp-pip and the side-chain carboxylic acid of Asp was
synthesized by the already described method. After cyclization, a
residue formed by concentration under reduced pressure was
dissolved in dichloromethane, then this was aliquoted into 10 test
tubes, and then they were concentrated again by removing the
solvent under reduced pressure.
[0491] To one of the 10 test tubes aliquoted, 0.40 mL of 0.05 M
tetramethylammonium hydrogen sulfate/HFIP solution (2% TIPS) (34.3
mg of tetramethylammonium hydrogen sulfate was dissolved in 4 mL of
solution drawn out from a solution produced by mixing HFIP: 11.66
mL, TIPS: 0.24 mL, and DCE: 0.10 mL) was added. The test tube was
capped using a rubber septum, shaken for three minutes, and then
left to stand at 25.degree. C. At the stage of four hours, the
reaction was checked by LCMS (SQDFA05). As a result, completion of
side-chain deprotection (deprotection of the DMT group of
MeSer(DMT) and deprotection of the Trt group of Set(Trt)) could be
confirmed. Here the UV area ratio according to LC of the
deprotected desired peptide (Compound 133; cyclic compound in which
an amide bond was formed between the N-terminal amino group of
H-g-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip
and the side-chain carboxylic acid of Asp) and the N- to O-acyl
shifted product of the desired peptide (depsipeptide) was 96:4.
Taking LCMS (SQDFA05) analyses after leaving the reaction solution
to stand at 25.degree. C. for 22 hours from the start of the
reaction resulted in UV area ratio according to LC of the
deprotected desired peptide (Compound 133; cyclic compound in which
an amide bond was formed between the N-terminal amino group of
H-g-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip
and the side-chain carboxylic acid of Asp) and the N- to O-acyl
shifted product of the desired peptide (depsipeptide) of 83:17
(FIG. 4).
[0492] The data of FIG. 4 are shown below: [0493] Desired peptide
(Compound 133; cyclic compound in which an amide bond was formed
between the N-terminal amino group of
H-g-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-CD-Ser-Trp-Trp-Pro-MeGly-Asp-pip
and the side-chain carboxylic acid of Asp) [0494] LCMS (ESI)
m/z=1474.1 (M+H)+ [0495] Retention time: 0.78 minutes (analysis
condition SQDFA05) [0496] Product of N- to O-acyl shift [0497] LCMS
(ESI) m/z=1474.1 (M+H)+ [0498] Retention time: 0.64 minutes
(analysis condition SQDFA05)
[0499] As described later, when the cyclic compound (Compound 103,
Pep3) in which an amide bond was formed between the N-terminal
amino group of
H-g-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3-Cl)-Ser(Trt)-Trp-Trp-Pro-MeGly-A-
sp-pip and the side-chain carboxylic acid of Asp was deprotected
under conditions of 5% TFA/DCE, deprotection for two hours at
25.degree. C. resulted in 70% progress of the N- to O-acyl
shift,
[0500] On the other hand, by using 0.05 M tetramethylammonium
hydrogen sulfate/HFIP solution (2% TIPS) instead of 5% TFA on the
same peptide sequence, generation of N- to O-acyl shift products
could be reduced significantly.
Example 3-2-4
Deprotection of the Cyclic Compound (Compound 101, Pep1) in which
an Amide Bond was Formed between the N-Terminal Amino Group of
H-Ala-Trp-Nle-Trp-D-Tyr(tBu)-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip
and the Side-Chain Carboxylic Acid of Asp, by Using 0.05 M oxalic
acid/HFIP Solution (2% TIPS) as the Deprotection Condition
[0501] After synthesizing Compound 101 (Pep1), 0.40 mL of 0.05 M
oxalic acid/HFIP solution (2% TIPS) (18.0 mg of oxalic acid was
dissolved in 4 mL of solution drawn out from a solution produced by
mixing HFIP: 11.66 mL, TIPS: 0.24 mL, and DCE: 0.10 mL) was added
to another one of 10 test tubes aliquoted in the above-mentioned
operation. The test tube was capped using a rubber septum, shaken
for three minutes, and then left to stand at 25.degree. C. for four
hours, and then the reaction was checked by LCMS (FA05). As a
result, side-chain deprotection (deprotection of the tBu group of
D-Tyr(tBu)) was completed, and at this time, the ratio among the
deprotected desired peptide (Compound 131), the solvolysis product
(compound showing a mass in which any of the amide bonds of the
peptide has undergone solvolysis by the solvent HFIP), and the
hydrolysate (compound showing a mass in which any of the amide
bonds of the peptide has undergone hydrolysis by water) was 79:17:4
(FIG. 5). "TM+H2O" represents a compound in which any one of the
amide bonds of the target molecule has undergone hydrolysis.
Similarly, "TM+HFIP" represents a compound in which any one of the
amide bonds of the target molecule has undergone solvolysis by
HFIP.
[0502] The data of FIG. 5 are shown below: [0503] Desired peptide
(Compound 131) [0504] LCMS (ESI) m/z=1423.5 (M+H)+ [0505] Retention
time: 0.79 minutes (analysis condition SQDFA05) [0506] Hydrolysate
[0507] LCMS (ESI) m/z=1441.5 (M+H)+ [0508] Retention time: 0.61
minutes (analysis condition SQDFA05) [0509] Product of solvolysis
by HFIP [0510] LCMS (ESI) m/z=1591.5 (M+H)+ [0511] Retention time:
0.68 minutes, 0.71 minutes (analysis condition SQDFA05)
Example 3-2-5
Deprotection of Cyclic Compound (Compound 101, Pep1) in which an
Amide Bond was Formed Between the N-Terminal Amino Group of
H-Ala-Trp-Nle-Trp-D-Tyr(tBu)-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip
and the Side-Chain Carboxylic Acid of Asp, by Using 0.05 M Maleic
Acid/HFIP Solution (2% TIPS) as the Deprotection Condition
[0512] After synthesizing Compound 101 (Pep 1), 0.40 mL of 0.05 M
maleic acid/HFIP solution (2% TIPS) (23.2 mg of maleic acid was
dissolved in 4 mL of solution drawn out from a solution produced by
mixing HFIP: 11.66 mL, TIPS: 0.24 mL, and DCE: 0.10 mL) was added
to another one of 10 test tubes aliquoted in the above-mentioned
operation. The test tube was capped using a rubber septum, shaken
for three minutes, and then left to stand at 25.degree. C. for four
hours, and then the reaction was checked by LCMS (FA05). As a
result, side-chain deprotection (deprotection of the tBu group of
D-Tyr(tBu)) was completed, and at this time, the ratio among the
deprotected desired peptide (Compound 131), the solvolysis product
(compound showing a mass in which any of the amide bonds of the
peptide has undergone solvolysis by the solvent HFIP), and the
hydrolysate (compound showing a mass in which any of the amide
bonds of the peptide has undergone hydrolysis by water) was 81:12:7
(FIG. 6). "TM+H2O" represents a compound in which any one of the
amide bonds of the target molecule has undergone hydrolysis.
Similarly, "TM+HFIP" represents a compound in which any one of the
amide bonds of the target molecule has undergone solvolysis by
HFIP.
[0513] The data of FIG. 6 are shown below: [0514] Desired peptide
(Compound 131) [0515] LCMS (ESI) m/z=1423.5 (M+H).sup.+ [0516]
Retention time: 0.79 minutes (analysis condition SQDFA05) [0517]
Hydrolysate [0518] LCMS (ESI) m/z=1441.5 (M+H).sup.+ [0519]
Retention time: 0.61 minutes (analysis condition SQDFA05) [0520]
Product of solvolysis by HFIP [0521] LCMS (ESI) m/z=1591.4
(M+H).sup.+ [0522] Retention time: 0.68 minutes, 0.71 minutes
(analysis condition SQDFA05)
Example 3-2-6
Deprotection of the Cyclic Compound (Compound 103, Pep3) in which
an Amide Bond was Formed Between the N-Terminal Amino Group of
H-g-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3-Cl)-Ser(Trt)-Trp-Trp-Pro-MeGly-A-
sp-pip and the Side-Chain Carboxylic Acid of Asp, by Using 0.05 M
oxalic acid/HFIP Solution (2% TIPS) as the Deprotection
Condition
[0523] After synthesizing Compound 103 (Pep3). 0.40 mL of 0.05 M
oxalic acid/HFIP solution (2% TIPS) (18.0 mg of oxalic acid was
dissolved in 4 mL of solution drawn out from a solution produced by
mixing HFIP: 11.66 mL, TIPS: 0.24 mL, and DCE: 0.10 mL) was added
to another one of 10 test tubes aliquoted in the above-mentioned
operation. The test tube was capped using a rubber septum, shaken
for three minutes, and then left to stand at 25.degree. C. for four
hours, and then the reaction was checked by LCMS (SQDFA05). As a
result, completion of side-chain deprotection (deprotection of the
DMT group of MeSer(DMT) and deprotection of the Trt group of
Ser(Trt)) could be confirmed. At this time, the UV area ratio
according to LC of the deprotected desired peptide (Compound 133;
cyclic compound in which an amide bond was formed between the
N-terminal amino group of
H-g-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-As-
p-pip and the side-chain carboxylic acid of Asp) and the N- to
O-acyl shifted product of the desired peptide (depsipeptide) was
86:14 (FIG. 7).
[0524] The data of FIG. 7 are shown below: [0525] Desired peptide
(Compound 133; cyclic compound in which an amide bond was formed
between the N-terminal amino group of
H-g-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip
and the side-chain carboxylic acid of Asp) [0526] LCMS (ESI)
m/z=1473.5 (M+H)+ [0527] Retention time: 0.78 minutes (analysis
condition SQDFA05) [0528] Product of N- to O-acyl shift. [0529]
LCMS (ESI) m/z=1473.5 (M+H)+ [0530] Retention time: 0.64 minutes
(analysis condition SQDFA05)
Example 3-2-7
Deprotection of the Cyclic Compound (Compound 103, Pep3) in which
an Amide Bond was Formed Between the N-Terminal Amino Group of
H-g-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3-Cl)-Ser(Tn)-Trp-Trp-Pro-MeGly-As-
p-pip and the Side-Chain Carboxylic Acid of Asp, by Using 0.05 M
Maleic Acid/HFIP Solution (2% TIPS) as the Deprotection
Condition
[0531] After synthesizing Compound 103 (Pep3), 0.40 mL of 0.05 M
maleic acid/HFIP solution (2% TIPS) (23.2 mg of maleic acid was
dissolved in 4 mL of solution drawn out from a solution produced by
mixing HFIP: 11.66 mL, TIPS: 0.24 mL, and DCE: 0.10 mL) was added
to another one of 10 test tubes aliquoted in the above-mentioned
operation. The test tube was capped using a rubber septum, shaken
for three minutes, and then left to stand at 25.degree. C. for four
hours, and then the reaction was checked by LCMS (SQDFA05). As a
result, completion of side-chain deprotection (deprotection of the
DMT group of MeSer(DMT) and deprotection of the Trt group of
Ser(Trt)) could be confirmed. At this time, the UV area ratio
according to LC of the deprotected desired peptide (Compound 133;
cyclic compound in which an amide bond was formed between the
N-terminal amino group of
H-g-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-As-
p-pip and the side-chain carboxylic acid of Asp) and the N- to
O-acyl shifted product of the desired peptide (depsipeptide) was
86:14 (FIG. 8).
[0532] The data of FIG. 8 are shown below: [0533] Desired peptide
(Compound 133; cyclic compound in which an amide bond was formed
between the N-terminal amino group of
H-g-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip
and the side-chain carboxylic acid of Asp) [0534] LCMS (ESI)
m/z=1473.5 (M+H)+ [0535] Retention time: 0.79 minutes (analysis
condition SQDFA05) [0536] Product of N- to O-acyl shift [0537] LCMS
(ESI) m/z=1473.5 (M+H)+ [0538] Retention time: 0.64 minutes
(analysis condition SQDFA05)
[0539] These results showed that even when oxalic acid (pKa 1.23)
or maleic acid (pKa 1.92) is used instead of tetramethylammonium
hydrogen sulfate (pKa 2.0) as the weak acid, deprotection can be
made to proceed while suppressing hydrolysis, solvolysis, and N- to
O-acyl shift.
Example 3-2-8
Comparison Between the Use of 0.05 M tetramethylammonium hydrogen
sulfate/HFIP (2% TIPS) as the Deprotection Condition and the Use of
0.05 M tetramethylammonium hydrogen sulfate/TFE (2% TIPS) as the
Deprotection Condition in the Deprotection of the Cyclic Compound
(Compound 107, Pep7) in which an Amide Bond was Formed Between the
N-Terminal Amino Group of
H-Ala-Phe(4-CF3)-Trp-Trp-MeLeu-MeGly-MeGly-Pro-Hyp(Et)-Ser(Trt)-Asp-pip(t-
Bu) and the Side-Chain Carboxylic Acid of Asp
[0540] Using Fmoc-Asp(O-Trt(2-Cl)-resin)-piptBu (Compound 52,
loading: 0.356 mmol/g, 100 mg) as the resin, cyclic compound
(Compound 107) in which an amide bond was formed between the
N-terminal amino group of
H-Ala-Phe(4-CF3)-Trp-Trp-MeLeu-MeGly-MeGly-Pro-Hyp(Et)-Ser(Trt)-Asp-pip(t-
Bu) and the side-chain carboxylic acid of Asp was synthesized by
the already described method. After cyclization, a residue produced
by concentration wider reduced pressure was dissolved in
dichloromethane, then this was aliquoted into 10 test tubes, and
then the solvent was distilled off again under reduced
pressure.
[0541] To one of the 10 test tubes aliquoted, 0.40 mL of 0.05 M
tetramethylammonium hydrogen sulfate/HFIP solution (2% TIPS) (a
solution produced by dissolving 205.8 mg of tetramethylammonium
hydrogen sulfate in a solution produced by mixing HFIP: 23.32 mL,
TIPS: 0.48 mL, and DCE: 0.20 mL) was added. The test tube was
capped using a rubber septum, and then left to stand at 25.degree.
C. for four hours, and the reaction was checked by LCMS (FA05). As
a result, side-chain deprotection (deprotection of the Trt group of
Ser(Trt)) was found to be completed, and at this point, the UV area
ratio between the deprotected desired peptide (Compound 137) and
the solvolysis product (compound showing a mass in which any of the
amide bonds of the peptide has undergone solvolysis by the solvent
HFIP) was 53:47 (FIG. 9), "TM+HFIP" represents a compound in which
any one of the amide bonds of the target molecule has undergone
solvolysis by HFIP.
##STR00068##
[0542] The data of FIG. 9 are shown below: [0543] Desired peptide
(Compound 137) [0544] LCMS (ESI) m/z=1492.1 (M+H)+ [0545] Retention
time: 0.90 minutes (analysis condition SQDRA05) [0546] Product of
solvolysis by HFIP (product in which any one of the amide bonds has
undergone solvolysis by HFIP) [0547] LCMS (ESI) m/z=1660.1 (M+H)+
[0548] Retention time: 0.78 minutes (analysis condition
SQDFA05)
[0549] To another test tube of 10 test tubes aliquoted, 0.40 mL of
0.05 M tetramethylammonium hydrogen sulfate/TFE solution (2% TIPS)
(a solution produced by dissolving 205.8 mg of tetramethylammonium
hydrogen sulfate in a solution produced by mixing TFE: 23.32 mL,
TIPS: 0.48 mL, and DCE: 0.20 mL) was added. The test tube was
capped using a rubber septum, and then left to stand at 25.degree.
C., and the reaction was checked by LCMS (FA05). As a result, four
hours later, the side-chain deprotection (deprotection of the Trt
group of Ser(Trt)) had proceeded 96%, and at this time, the
solvolysis product (compound showing a mass in which any of the
amide bonds of the peptide has undergone solvolysis by the solvent
TFE) was detection limit or below by LCMS. When the reaction was
checked 20 hours later, the side-chain deprotection (deprotection
of the Trt group of Ser(Trt)) completed, and at this time, the UV
area ratio of the deprotected desired peptide (Compound 137) and
the solvolysis product (compound showing a mass in which any of the
amide bonds of the peptide has undergone solvolysis by the solvent
TFE) was 97:3 (FIG. 10). "TM+TFE" recited in this Example
represents a compound in which the target molecule (TM) has
undergone solvolysis by TFE (a compound in which any one of the
amide bonds has undergone solvolysis by TFE).
[0550] The data of FIG. 10 are shown below: [0551] Desired peptide
(Compound 137) [0552] LCMS (ESI) m/z=1492.2 (M+H)+ [0553] Retention
time: 0.90 minutes analysis condition SQDFA05) [0554] Product of
solvolysis by TFE (product in which any of the amide bonds has
undergone solvolysis by TFE) [0555] LCMS (ESI) m/z=1592.0 (M+H)+
[0556] Retention time: 075 minutes (analysis condition SQDFA05)
[0557] The above-mentioned results showed that TFE can be used
instead of HFIP as the solvent for dissolving the weak acid. These
results also suggest the possibility that any solvent may be used
as long as the following conditions are met: YOTs value is
positive, the solvent itself is weakly acidic (aqueous pKa of 5 to
14) and nucleophilicity is low.
Example 3-2-9
Deprotection Using 0.1 M tetramethylammonium hydrogen sulfate/HFIP
Solution (2% TIPS) as the Deprotection Condition, and Stopping the
Reaction by Adding a Base (DIPEA) to this Solution
[0558] Using Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (Compound 50, loading:
0.342 mmol/g, 100 mg) as the resin, the cyclic compound (Compound
105, Pep5) in which an amide bond was formed between the N-terminal
amino group of
H-Ala-Trp-Nle-Trp-Ser(Trt)-Gly-MeAla-MePhe(3-Cl)-MeGly-Pro-Asp-pip
and the side-chain carboxylic acid of Asp was synthesized by the
already described method. After cyclization, a residue produced by
concentration under reduced pressure was dissolved in
dichloromethane, then this was aliquoted into 10 test tubes, and
then they were concentrated again by removing the solvent under
reduced pressure.
[0559] To two of the 10 test tubes aliquoted, 0.40 mL of 0.1 M
tetramethylammonium hydrogen sulfate/HFIP solution (2% TIPS) (68.5
mg of tetramethylammonium hydrogen sulfate was dissolved in 4 mL of
solution drawn out from a solution produced by mixing HFIP: 11.66
mL, TIPS: 0.24 mL, and DCE: 0.10 mL) was added, respectively. The
test tubes were capped respectively using a rubber septum, shaken
for three minutes, and then left to stand at 25.degree. C. for four
hours, and the reactions were checked by LCMS (SQDFA05). As a
result, side-chain deprotection (deprotection of the Trt group of
Ser(Trt)) was completed, and at this time, peaks showing the masses
of compounds other than the deprotected desired peptide (Compound
135), which are the solvolysis product (compound showing a mass in
which any of the amide bonds of the peptide has undergone
solvolysis by the solvent HFIP) and hydrolysate (compound showing a
mass in which any of the amide bonds of the peptide was undergone
solvolysis by water), were not detected (FIG. 11). To each of these
reaction mixtures, diisopropylethylamine (DIPEA, 14 .mu.L, two
equivalents with respect to tetramethylammonium hydrogen sulfate)
was added, and one of the test tubes was left to stand at
25.degree. C. for 18 hours, the other test tube was subjected to
concentration under reduced pressure. When their LCMS (SQDFA05)
analyses were taken, peaks showing the masses of compounds other
than the deprotected desired peptide, which are the solvolysis
product and the hydrolysate, were not detected at this point as
well (FIG. 11).
##STR00069##
[0560] The data of FIG. 11 are shown below: [0561] Desired peptide
(Compound 135) [0562] LCMS (ESI) m/z=1331.9 (M+H)+ [0563] Retention
time: 0.74 minutes (analysis condition SQDFA05)
Example 3-2-10
Deprotection Using 0.1 M tetramethylammonium hydrogen sulfate/HFIP
Solution (2% TIPS) as the Deprotection Condition, and Stopping the
Reaction by Adding a Base (DIPEA) to this Solution
[0564] Using Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (Compound 50, loading:
0.316 mmol/g, 100 mg) as the resin, the cyclic compound (Compound
103, Pep3) in which an amide bond was formed bet the N-terminal
amino group of
H-g-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3-Cl)-Ser(Trt)-Trp-Trp-Pro-MeGly-A-
sp-pip and the side-chain carboxylic acid of Asp was synthesized by
the already described method. After cyclization, a residue produced
by concentration under reduced pressure was dissolved in
dichloromethane, then this was aliquoted into 10 test tubes, and
then they were concentrated again by removing the solvent under
reduced pressure.
[0565] To two of the 10 test tubes aliquoted, 0.40 mL of 0.1 M
tetramethylammonium hydrogen sulfate/HFIP solution (2% TIPS) (68.5
mg of tetramethylammonium hydrogen sulfate was dissolved in 4 mL of
solution drawn out from a solution produced by mixing HFIP: 11.66
mL, TIPS: 0.24 mL, and DCE: 0.10 mL) was added, respectively. The
test tubes were capped respectively using a rubber septum, shaken
for three minutes, and then left to stand at 25.degree. C. for four
hours, and the reactions were checked by LCMS (FA05). As a result,
side-chain deprotections (deprotection of the DMT group of
MeSer(DMT) and deprotection of the Trt group of Ser(Trt)) were
completed, and the UV area ratio according to LC of the deprotected
desired peptide (Compound 133) and the N- to O-acyl shifted product
of the desired peptide (depsipeptide) was 93:7.
[0566] To each of these reaction mixtures, diisopropylethylamine
(DIPEA, 14 .mu.L, two equivalents with respect to
tetramethylammonium hydrogen sulfate) was added, and one of the
test tubes was left to stand at 25.degree. C. for 18 hours, the
other test tube was subjected to concentration under reduced
pressure immediately after DIPEA addition. When their LCMS (FA05)
analyses were taken, the mixture left to stand for 18 hours did not
show change in the UV area ratio of the desired peptide to the N-
to O-acyl shift product thereof; whereas the mixture which was
concentrated under reduced pressure had a UV area ratio of the
desired peptide to the N- to O-acyl shift product thereof of 98:2
(FIG. 12).
[0567] The data of FIG. 12 are shown below: [0568] Desired peptide
(Compound 133) [0569] LCMS (ESI) m/z=1474.1 (M+H)+ [0570] Retention
time: 0.78 minutes (analysis condition SQDFA05) [0571] N- to O-acyl
shift product [0572] LCMS (ESI) m/z=1474.1 (M+H)+ [0573] Retention
time: 0.64 minutes analysis condition SQDFA05)
[0574] The above-mentioned results showed that work-up is possible
under conditions where the problems of hydrolysis (solvolysis) and
N- to O-acyl shift are suppressed, by adding DIPEA after the
deprotection reaction has been completed (or when one wants to stop
the reaction)
Example 4
Reactivity of Thr and MeSer when Using THP Group as the Protecting
Group for the Side-Chain Hydroxyl Groups
[0575] The following experiments were performed for the reactivity
when using THP group as the protecting group for the side-chain
hydroxyl groups of Thr and MeSer for which low reactivity during
the elongation reaction is of concern.
Example 4-1
Comparative Evaluation of the Elongation Reactivity of
Fmoc-Thr(Trt)-OH and Fmoc-Thr(rHP)-OH (Compound 2) on the Compound
having an N-methylamino Group at its N-terminus
(H-MePhe(3-Cl)-D-Tyr(tBu)-Trp-MePhe-Trp-MePhe-Ile-Asp(O-2-Cl-Trt-resin)-p-
ip), which is a Peptide Elongated on a Resin
[0576] Comparative evaluation of elongation reactivity was
performed using a sequence having MePhe(3-Cl) at its N-terminus,
which has low amino group reactivity due to steric hindrance.
[0577]
Fmoc-MePhe(3-Cl)-D-Tyr(tBu)-Trp-MePhe-Trp-MePhe-Ile-Asp(O-Trt(2-Cl)-
-resin)-pip (Compound 108) was synthesized by the already described
method, by using as the resin Fmoc-Asp(O-Trt(2-Cl)-resin)-pip
(Compound 50, loading: 0.329 mmol/g, 100 g) prepared by the already
described method.
[0578] To the obtained
Fmoc-MePhe(3-Cl)-D-Tyr(tBu)-Trp-MePhe-Trp-MePhe-Ile-Asp(O-Trt(2-Cl)-resin-
)-pip (Compound 108), dichloromethane (600 .mu.L) was added, and
this was left to stand for 30 minutes to allow swelling of the
resin. After removing the liquid phase, the resin was washed three
times with DMF (600 .mu.L). To the obtained resin, 2% DBU/DMF (v/v,
600 .mu.L) was added, this was shaken for 20 minutes, and the
liquid phase was removed. The resin was washed three times with DMF
(600 .mu.L).
[0579] To this resin, a solution produced by mixing a solution of
0.60 M Fmoc-Thr(Trt)-OH/0.375 M oxyma in NMP (300 .mu.L) and 10%
(v/v) DIC/DMF (300 .mu.L), or a solution produced by mixing a
solution of 0.60 M Fmoc-Thr(THP)-OH (Compound 2)/0.375 M oxyma in
NMP (300 .mu.L) and 10% (v/v) DIC/DMF (300 .mu.L) was added, and
this was shaken. While shaking, approximately 10 mg of the reacting
resin was collected at the stages of shaking for one hour, two
hours, and four hours, respectively, the collected resins were
washed three times with DMF (600 .mu.L), 2% DBU/DMF (v/v, 600
.mu.L) was additionally added and this was shaken for 20 minutes,
and the liquid phase was removed. The resins were washed three
times with DMF (600 .mu.L), and then three times with
dichloromethane (600 .mu.L).
[0580] TFE/DCM (1/1, v/v, 1 mL) was added to the obtained resins,
these were shaken for ten minutes. After removing the resins
filtering, the liquid phases were concentrated under reduced
pressure. The residues were analyzed by LCMS (analysis condition
SQDFA05). The results are shown in Table 4.
TABLE-US-00006 TABLE 4 Elongated 1 h 2 h 4 h Amino (Unreacted Form/
(Unreacted Form/ (Unreacted Form/ Add Residue Elongated Form)
Elongated Form) Elongated Form) Thr (Trt) 99/1 97/3 88/12 Thr (THP)
84/16 69/31 22/78
[0581] The ratios of the unreacted form/elongated form in the Table
show the UV area ratios according to LC. In the Table, unreacted
form means H-MePhe(3-Cl)-D-Tyr(tBu)-Trp-MePhe-Trp-MePhe-Ile-Asp-pip
(Compound 109), and the elongated form means
H-Thr(Trt)-MePhe(3-Cl)-D-Tyr(tBu)-Trp-MePhe-Trp-MePhe-Ile-Asp-pip
(Compound 110; when Fmoc-Thr(Trt)-OH was added) or
H-Thr(THP)-MePhe(3-Cl)-D-Tyr(tBu)-Trp-MePhe-Trp-MePhe-Ile-Asp-pip
(Compound 111; when Fmoc-Thr(THP)-OH was added).
##STR00070##
[0582] Unreacted form (Compound 109)
[0583] LCMS (ESI) m/z=1422.9 (M+H)+
[0584] Retention time: 0.77 minutes (analysis condition
SQDFA05)
##STR00071##
[0585] Elongated form (Thr(Trt)) (Compound 110)
[0586] LCMS (ESI) m/z=1766.2 (M+H)+
[0587] Retention time: 0.92 minutes (analysis condition
SQDFA05)
##STR00072##
[0588] Elongated form (Thr(THP)) (Compound 111)
[0589] LCMS (ESI) m/z=1608.1 (M+H)+
[0590] Retention time: 0.80 minutes (analysis condition
SQDFA05)
Example 4-2
Elongation Reactivity of Fmoc-Thr(Trt)-OH or Fmoc-Thr(THP)-OH
(Compound 2) when Synthesizing
H-b-MeAla-Ile-MeLeu-MeAla-MeLeu-Thr(PG)-MePhe-MeAla-MeLeu-MePhe-Asp(O-Trt-
(2-Cl)-resin)-pip by a Synthesizer
[0591] Elongation reactivity was tested by elongating Thr to a
sequence having MePhe at its N terminus, which has low reactivity
due to steric hindrance, and to a sequence having MeLeu, which is
sterically bulky with respect to the amino group of Thr, at its N
terminus.
[0592] Peptide elongation of
H-b-MeAla-Ile-MeLeu-MeAla-MeLeu-Thr(PG)-MePhe-MeAla-MeLeu-MePhe-Asp(O-Trt-
(2-Cl)-resin)-pip (wherein, PG on the Thr side chain represents a
protecting group, and in this experiment, it represents protection
by Trt or THP) was performed according to peptide synthesis method
using the Fmoc method already described in the Examples, by using
as the resin Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (Compound 50, 100 mg)
prepared by the already described method. In this case,
Fmoc-Thr(Trt)-OH or Fmoc-Thr(THP)-OH (Compound 2) was used in the
Thr elongation.
[0593] After the peptide elongation, removal of the N-terminal Fmoc
group was performed on the peptide synthesizer, and the resin was
washed with DMF and DCM.
[0594] After swelling the resin again with DCM, TFE/DCM (1/1, v/v,
2 mL) was added to the resin, this was shaken at room temperature
for two hours, and the peptides were cleaved off from the resin.
Next, the resin was removed by filtering the solution inside the
tube through a column for synthesis, and the remaining resin was
further washed twice with TFE/DCM (1/1, v/v, 1 mL).
[0595] After the cleavage, when using Fmoc-Thr(Trt)-OH, the Trt
group of Thr(Trt) was deprotected by using a solution produced by
mixing 4N HCl/1,4-dioxane (19.5 .mu.L), TIPS (0.25 mL), and DCM
(0.73 mL) and the mixture was shaken at 25.degree. C. for five
minutes. Then acid was neutralized by adding DIPEA (24 .mu.L), and
the elongation reactivity was tested by LCMS.
[0596] The LCMS results for the products are shown in FIG. 13. The
desired peptide (Compound 112,
H-b-MeAla-Ile-MeLeu-MeAla-MeLeu-Thr-MePhe-MeAla-MeLeu-MePhe-Asp-pip)
and the compound in which Thr was lost from the desired peptide
(Compound 113, H-b-MeAla-Ile-MeLeu-MeAla-MeLeu-
MePhe-MeAla-MeLeu-MePhe-Asp-pip) were observed at the same
retention time of 0.69 minutes. Furthermore, according to MS
(negative mode), Thr-lost peptide was included at a proportion of
approximately 30%.
##STR00073##
[0597] The data of FIG. 13 are shown below: [0598] Target molecule
(Compound 112,
H-b-MeAla-Ile-MeLeu-MeAla-MeLeu-Thr-MePhe-MeAla-MeLeu-MePhe-Asp-pip)
[0599] LCMS (ESI) m/z=1373.6 (M+H)+ [0600] Retention time: 0.69
minutes (analysis condition SQDAA50)
##STR00074##
[0601] Target molecule--Thr (Compound 113,
H-b-MeAla-Ile-MeLeu-MeAla-MeLeu-MePhe-MeAla-MeLeu-MePhe-Asp-pip)
[0602] LCMS (ESI) m/z=1272.6 (M+H)+ [0603] Retention time: 0.69
minutes (analysis condition SQDAA50)
[0604] The LCMS results for this product are shown in FIG. 14. In
contrast to the case when Fmoc-Thr(Trt)-OH was added, when
Fmoc-Thr(THP)-OH was added, the peptide (Compound 113,
H-b-MeAla-Ile-MeLeu-MeAla-MeLeu-MePhe-MeAla-MeLeu-MePhe-Asp-pip) in
which Thr was lost from the desired peptide (Compound 114,
H-b-MeAla-IIe-MeLeu-MeAla-MeLeu-Thr(THP)-MePhe-MeAla-MeLeu-MePhe-Asp-pip)
was not detected. When using Fmoc-Thr(THP)-OH (Compound 2),
deprotection operation was not performed after cleavage from the
resin, and the cleavage solution was directly subjected to LCMS
analyses; therefore, the THP protection of the Thr side chain
remained.
##STR00075##
[0605] The data of FIG. 14 are shown below: [0606] Target molecule
(Compound 114) [0607] LCMS (ESI) m/z=1458.1 (M+H)+ [0608] Retention
time: 0.73 minutes (analysis condition SQDFA05)
[0609] The above-mentioned results showed that compared to
Fmoc-Thr(Trt)-OH used for Thr elongation in standard peptide
synthesis, Fmoc-Thr(THP)-OH (Compound 2) has higher reactivity. In
particular, when elongating from a bulky amino group of an
N-alkylated N terminus, high condensation efficiency was shown to
be achievable. Furthermore, in the subsequent elongation reaction,
it can be confirmed that elongation of an Fmoc-amino acid
(Fmoc-MeLeu-OH in this case), which is bulky with respect to the
N-terminal amino group of Thr(THP), proceeds without any
problem.
Example 4-2
Confirmation of Elongation Reactivity of MeSer when Using
Fmoc-MeSer(DMT)-OH or Fmoc-MeSer(THP)-OH (Compound 6) in the
Synthesis of
H-MeSer(PG)-MeVal-MeHis(Trt)-Tyr(3-F,tBu)-Pro-MeHis(Trt)-Pro-Trp-MePhe(4--
Cl)-Asp(O-Th(2-Cl)-resin)-Pro-OPis by a Synthesizer
[0610] Peptide elongation of
H-MeSer(PG)-MeVal-MeHis(Trt)-Tyr(3-F,tBu)-Pro-MeHis(Trt)-Pro-Trp-MePhe(4--
Cl)-Asp(O-Trt(2-Cl)-resin)-Pro-OPis (wherein PG on the MeSer side
chain represents a protecting group, and in this experiment, it
represents protection by DMT or THP) was performed according to the
peptide synthesis method using the Fmoc method already described in
the Examples, by using as the resin
Fmoc-Asp(O-Trt(2-Cl)-resin)-Pro-OPis (Compound 58, loading rate:
0.3736 mmol/g, 100 mg) prepared by the already described method. In
this case, Fmoc-MeSer(DMT)-OH or Fmoc-MeSer(THP)-OH (Compound 6)
was used in the MeSer elongation.
[0611] After the peptide elongation, N-terminal Fmoc group was
removed on the peptide synthesizer, and the resin was washed using
DMF and DCM.
[0612] After drying the obtained resin under reduced pressure, 30
mg each of the respective resins was collected. Each of the
collected 30 mg of resin was swollen again with DCM, then TFE/DCM
(1/1, v/v, 2 mL) was added to the resin, this was shaken at room
temperature for two hours, and the peptides were cleaved off from
the resin. Next, the resin was removed by filtering the solution
inside the tube through a column for synthesis, and the remaining
resin was further washed twice with TFE/DCM (1/1, v/v, 1 mL). All
of the obtained cleavage solutions were mixed, and concentrated
under reduced pressure.
[0613] To the obtained residue, 1.3 mL of 0.05 M
tetramethylammonium hydrogen sulfate/HFIP solution (2% TIPS)
prepared by the already described method was added to dissolve the
residue, and then this was left to stand at room temperature for
one hour. The side chain protecting groups other than the tBu
protection of the Tyr(3-F) side chain (DMT or THP protection of the
MeSer side chain and Trt protection of the Me His side chain) and
main-chain C-terminal Pis protection were deprotected, and the
reactions were checked by taking LCMS analyses.
[0614] FIG. 15 shows the results of LCMS when the synthesis is
carried out using Fmoc-MeSer(DMT)-OH0.75 DIPEA. The desired peptide
(Compound 115,
H-MeSer-MeVal-MeHis-Tyr(3-F,tBu)-Pro-MeHis-Pro-Trp-MePhe(4-Cl)-Asp-Pro-OH-
) and the compound in which MeSer was lost from the desired peptide
(Compound 116,
H-MeVal-MeHis-Tyr(3-F,tBu)-Pro-MeHis-Pro-Trp-MePhe(4-Cl)-Asp-Pro-OH)
were observed at the same retention time of 0.50 minutes.
[0615] Furthermore, according to MS (negative mode), MeSer-lost
peptide (Compound 116,
H-MeVal-MeHis-Tyr(3-F,tBu)-Pro-MeHis-Pro-Trp-MePhe(4-Cl)-Asp-Pro-OH)
was found to be included at a proportion of 50%.
##STR00076##
[0616] The data of FIG. 15 are shown below: [0617] Target molecule
(Compound 115) [0618] LCMS (ESI) m/z=1559.7 (M+H)+ [0619] Retention
time: 0.50 minutes (analysis condition SQDFA50) [0620] Target
molecule--MeSer (Compound 116) [0621] LCMS (ESI) m/z=1458.8 (M+H)+
[0622] Retention time: 0.50 minutes (analysis condition
SQDFA50)
##STR00077##
[0623] In contrast, FIG. 16 shows the results of LCMS when the
synthesis was carried out using Fmoc-MeSer(THP)-OH (Compound 6).
The desired peptide (Compound 115,
H-MeSer-MeVal-MeHis-Tyr(3-F,tBu)-Pro-MeHis-Pro-Trp-MePhe(4-Cl)-Asp-Pro-OH-
) and the compound in which MeSer was lost from the desired peptide
(Compound 116) were observed at the same retention time of 0.50
minutes. However, according to MS (negative mode), the MeSer-lost
peptide (Compound 116,
H-MeVal-MeHis-Tyr(3-F,tBu)-Pro-MeHis-Pro-Trp-MePhe(4-Cl)-Asp-Pro-OH)
was found to be included at a proportion of 10% or less.
[0624] The data of FIG. 16 are shown below: [0625] Target molecule
(Compound 115) [0626] LCMS (ESI) m/z=1559.7 (M+H)+ [0627] Retention
time: 0.50 minutes (analysis condition SQDFA50) [0628] Desired
peptide--MeSer (Compound 116) [0629] LCMS (ESI) m/z=1458.7 (M+H)+
[0630] Retention time: 0.50 minutes (analysis condition
SQDFA50)
[0631] The above-mentioned results showed that compared to
Fmoc-MeSer(DMT)-OH, Fmoc-MeSer(THP)-OH (Compound 6) has higher
reactivity and can achieve high condensation efficiency when
performing elongation from an amino group of an N-methylated N
terminus.
5. Deprotection of Cyclized Peptides in 5% TFA (Comparative
Examples)
Comparative Example 1
Deprotection of the Side-Chain Protecting Group (Deprotection of
the tBu Protection of D-Tyr(tBu)) of the Compound (Compound 101,
Pep-1) in which an Amide Bond was Formed Between the N-Terminal
Amino Group of
H-Ala-Trp-Nle-Trp-D-Tyr(tBu)-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip
and the Side-Chain Carboxylic Acid of Asp (when Using 5% TFA/DCE
(5% TIPS))
[0632] Using Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (Compound 50, resin
loading rate: 0.373 mmol/g, 100 mg) synthesized by the already
described method, peptide elongation was performed on a synthesizer
to obtain
H-Ala-Trp-Nle-Trp-D-Tyr(tBu)-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp(O-T-
rt(2-Cl)-resin)-pip.
[0633] Then, after swelling the resin again with DCM, TFE/DCM (1/1,
v/v, 2 mL) was added to the resin, this was shaken at room
temperature for two hours, and then the peptide was cleaved off
from the resin. Next, the resin was removed by filtering the
solution inside the tube through a column for synthesis, and the
remaining resin was further washed twice with TFE/DCM (1/1, v/v, 1
mL). All of the obtained cleavage solutions were mixed and
concentrated under reduced pressure.
[0634] The obtained residue was dissolved in DMF/DCM (1/1, v/v, 8
mL), a O-(7-aza-1H-benzotriazol-1-yl)-N,N,N,N-tetramethyluronium
hexafluorophosphate (HATU, 21 mg)/DME solution (0.5 M) and a DIPEA
(12 .mu.L)/DMF (88 .mu.L) solution were added, and this was stirred
at room temperature for two hours. The reaction was checked by LCMS
analyses (analysis condition SQDFA05), and generation of the
compound (Compound 101, Pep-1) in which an amide bond was formed
between the N-terminal amino group of
H-Ala-Trp-Nle-Trp-D-Tyr(tBu)-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip
and the side-chain carboxylic acid of Asp was confirmed. [0635]
LCMS (ESI) m/z=1480.0 (M+H).sup.+ [0636] Retention time: 0.93
minutes (analysis condition SQDEA05)
[0637] Thereafter, the solvent was distilled off under reduced
pressure, 5% TFA/DCE (5% Tips) (8 mL, moisture content was
confirmed to be <200 ppm by the Karl Fischer titration) was
added to the obtained residue, and this was stirred for 2.5 hours.
The solvent was distilled off under reduced pressure, and when the
residue was subjected to LCMS (FA05) analysis, masses corresponding
to the compound (Compound 131) in which an amide bond was formed
between the N-terminal amino group of
H-Ala-Trp-Nle-Trp-D-Tyr-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip
and the side-chain carboxylic acid of Asp, which is a target
molecule, and the hydrolysate thereof (product in which any of the
amide bonds was hydrolyzed) were confirmed, and the UV area ratio
corresponding to them was 13:87 (FIG. 17). The LC analysis results
are shown in FIG. 17. "TM+H2O" in this Example represents a
compound in which any one of the amide bonds of the target molecule
has undergone hydrolysis.
##STR00078##
[0638] The data of FIG. 17 are shown below:
[0639] Target molecule (Compound 131, compound in which an amide
bond was formed between the N-terminal amino group of
H-Ala-Trp-Nle-Trp-D-Tyr-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip
and the side-chain carboxylic acid of Asp) [0640] LCMS (ESI)
m/z=1424.0 (M+H)+ [0641] Retention time: 0.79 minutes (analysis
condition SQDFA05) [0642] Hydrolysate (product in which any of the
amide bonds of a compound has undergone hydrolysis, the compound in
which an amide bond was formed between the N-terminal amino group
of
H-Ala-Trp-Nle-Trp-D-Tyr-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip
and the carboxylic acid of the Asp side chain) [0643] LCMS (ESI)
m/z=1442.0 (M+H)+ [0644] Retention time: 0.61 minutes (analysis
condition SQDFA05)
[0645] These results confirmed that in deprotection using 5%
TFA/DCE (5% TIPS), in case of cyclic peptides having sequences that
are highly N-methylated, particularly sequences with consecutive
N-methyl amino acids, approximately 90% of the target molecule is
hydrolyzed, and obtaining the target molecule becomes
difficult.
Comparative Example 2
Deprotection of the Side-Chain Protecting Groups (DMT Protection of
MeSer Side Chain and Trt Protection of the Ser Side Chain) of the
Compound (Compound 103, Pep3) in which an Amide Bond was Formed
Between the N-Terminal Amino Group of
H-g-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3-Cl)-Ser(Trt)-Trp-Trp-Pro-MeGly-A-
sp-pip and the side-chain carboxylic acid of Asp (when using 5%
TFA/DCE (5% TIPS))
[0646] Using Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (Compound 50, resin
loading rate: 0.316 mmol/g, 100 mg) synthesized by the already
described method, peptide elongation was performed on a synthesizer
to obtain
H-g-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3-Cl)-Ser(Trt)-Trp-Trp-Pro-MeGly-A-
sp(O-Trt(2-Cl)-resin)-pip.
[0647] Then, after swelling the resin again with DCM, TFE/DCM (1/1,
v/v, 2 mL) was added to the resin, this was shaken at room
temperature for two hours, and then the peptide was cleaved off
from the resin. Next, the resin was removed by filtering the
solution inside the tube through a column for synthesis, and the
remaining resin was further washed twice with TFE/DCM (1/1, v/v, 1
mL).
[0648] The operation of cleaving the peptides off from the resin
was repeated twice, and all of the obtained cleavage solutions were
mixed and then concentrated under reduced pressure.
[0649] The obtained residue was dissolved in DMF/DCM (1/1, v/v, 8
mL), a O-(7-aza-1H-benzotriazol-1-yl)-N,N,N,N-tetramethyluronium
hexafluorophosphate (HATU, 18 mg)/DMF solution (0.5 M) and a DIPEA
(9.9 .mu.L)/DMF (39.6 .mu.L) solution were added, and this was
stirred at room temperature for two hours. The reaction was checked
by LCMS analyses (SQDFA05), and the following compounds were
confirmed: 70% of a compound formed by removal of DMT protection of
the MeSer side chain from the compound (Compound 103, Pep3) in
which an amide bond was formed between the N-terminal amino group
of
H-g-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3-Cl)-Ser(Trt)-Trp-Trp-Pro-MeGly-A-
sp-pip and the side-chain carboxylic acid of Asp (LCMS (ESI)
m/z=1716.2 (M+H)+; retention time: 1.06 minutes) and 30% of a
compound in which both DMT protection of the MeSer side chain and
Trt protection of the Ser side chain were removed from Compound 103
(LCMS (ESI) m/z=1474.1 (M+H).sup.+; retention time: 0.78 minutes).
The percentage was calculated from the UV area ratio according to
LC. Thereafter, the solvent was distilled off under reduced
pressure, the residue was dissolved in dichloromethane, and this
was aliquoted into 10 test tubes. These were concentrated under
reduced pressure to remove dichloromethane. The "residue after
cyclization" used in the above-mentioned Examples refers to a
residue obtained by concentration under reduced pressure after
aliquoting it into 10 tubes.
[0650] To one of these test tubes, 5% TFA/DCE (5% TIPS) (0.8 mL,
water content of 32.5 ppm determined by the Karl Fischer titration)
was added and shaken for three minutes, and then this was left to
stand at 25.degree. C.. for two hours. The solvent was distilled
off under reduced pressure, and when the residue was subjected to
LCMS (FA05) analysis, a compound (Compound 133) in which an amide
bond was formed between the N-terminal amino group of
H-g-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip
and the side-chain carboxylic acid of Asp, which is a target
molecule, the N- to O-acyl shift product of the target molecule, a
product in which one of the hydroxyl groups of the target molecule
was esterified with TFA, and a product in which two of the hydroxyl
groups of the target molecule were esterified with TFA were
confirmed to have a UV area ratio of 17:46:32:5. The LC analysis
results are shown in FIG. 18.
##STR00079##
[0651] The data of FIG. 18 are shown below: [0652] Desired peptide
(Compound 133, compound in which an amide bond was formed between
the N-terminal amino group of
H-g-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip
and the side-chain carboxylic acid of Asp) [0653] LCMS (ESI)
m/z=1473.8 (M+H)+ [0654] Retention time: 0.79 minutes (analysis
condition SQDFA05) [0655] N- to O-acyl shift product of the target
molecule (a compound in which N- to O-acyl shift progressed at
either one or both of the two hydroxyl groups of the target
molecule) [0656] LCMS (ESI) m/z=1473.8 (M+H)+ [0657] Retention
time: 0.62 minutes (analysis condition SQDFA05) [0658] Compound in
which one of the hydroxyl groups of the target molecule was
esterified with TFA [0659] LCMS (ESI) m/z=1569.8 (M+H)+ [0660]
Retention time: 0.89 minutes (analysis condition SQDFA05) [0661]
Compound in which two of the hydroxyl groups of the target molecule
were esterified with TFA [0662] LCMS (ESI) m/z=1665.9 (M+H)+ [0663]
Retention time: 0.99 minutes (analysis condition SQDFA05)
[0664] These results confirmed that when p-hydroxyl
group-containing amino acids such as MeSer and Ser are included in
a sequence, deprotection using 5% TFA/DCE (5% TIPS) causes N- to
O-acyl shift to progress. Furthermore, deprotection under this
condition was confirmed to cause TFA esterification to take place
on one or both of the two side-chain hydroxyl groups. It was
confirmed that obtaining the target molecule becomes difficult due
to these undesired reactions.
[0665] Examinations in which the reaction temperature is lowered to
0.degree. C., and examinations in which the TFA concentration is
lowered were carried out with the objective of suppressing
generation of N- to O-acyl shift products and suppressing TFA
esterification of hydroxyl groups.
Comparative Example
Deprotection of the Side-Chain Protecting Groups (DMT Protection of
the MeSer Side Chain and Trt Protection of the Ser Side Chain) of
the Compound (Compound 103, Pep3) in which an Amide Bond was Formed
Between the N-Terminal Amino Group of
H-g-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3-Cl)-Ser(Trt)-Trp-Trp-Pro-MeGly-A-
sp-pip and the Side-Chain Carboxylic Acid of Asp (when Performing
the Deprotection at 0.degree. C. using 5% TFA/DCE (5% TIPS))
[0666] After the above-mentioned cyclization, 5% TFA/DCE (5% TIPS)
(0.8 mL, moisture content of 36.6 ppm determined by the Karl
Fischer titration) was added at 0.degree. C. to one of the 10 test
tubes aliquoted, this was shaken for one minute, and then this was
left to stand at 0.degree. C. for four hours. When the reaction
solution was subjected to LCMS (FA05) analysis, the compound
(Compound 133) in which an amide bond was formed between the
N-terminal amino group of
H-g-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip
and the side-chain carboxylic acid of Asp, which is a target
molecule, the N- to O-acyl shift product of the target molecule, a
product in which one of the hydroxyl groups of the target molecule
was esterified with TFA, and a product in which one of the hydroxyl
groups of the N- to O-acyl shift product of the target molecule was
esterified with TFA were confirmed to have a UV area ratio of
56:12:21:11. The LC analysis results are shown in FIG. 19.
[0667] The data of FIG. 19 are shown below: [0668] Desired peptide
(Compound 133, compound in which an amide bond was formed between
the N-terminal amino group of
H-g-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip
and the side-chain carboxylic acid of Asp) [0669] LCMS (ESI)
m/z=1473.7 (M+H)+ [0670] Retention time: 0.78 minutes (analysis
condition SQDFA05) [0671] N- to O-acyl shift product of the target
molecule (a compound in which N- to O-acyl shift progressed at
either one or both of the two hydroxyl groups of the target
molecule) [0672] LCMS (ESI) m/z=1473.7 (M+H)+ [0673] Retention
time: 0.65 minutes (analysis condition SQDFA05) [0674] Compound in
which one of the hydroxyl groups of the target molecule was
esterified with TFA [0675] LCMS (ESI) m/z=1569.7 (M+H)+ [0676]
Retention time: 0.89 minutes (analysis condition SQDFA05) [0677]
Compound in which one of the hydroxyl groups of the N- to O-acyl
shift product of the target molecule was esterified with TFA [0678]
LCMS (ESI) m/z=1569.7 (M+H)+ [0679] Retention time: 0.73 minutes
(analysis condition SQDFA05)
Comparative Example 4
Deprotection of the Side-Chain Protecting Groups (DMT Protection of
the MeSer Side Chain and Trt Protection of the Ser Side Chain) of
the Compound (Compound 103, Pep3) in which an Amide Bond was Formed
Between the N-Terminal Amino Group of
H-g-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3-Cl)-Ser(Trt)-Trp-Trp-Pro-MeGly-A-
sp-pip and the Side-Chain Carboxylic Acid of Asp (when Performing
the Deprotection at 25.degree. C. and Using 2% TFA/DCE (5%
TIPS))
[0680] After the above-mentioned cyclization, 2% TFA/DCE (5% TIPS)
(0.8 mL, water content of 30.1 ppm determined by the Karl Fischer
titration) was added at 25.degree. C. to one of the 10 test tubes
aliquoted, this was shaken for one minute, and then this was left
to stand at room temperature for four hours. When the reaction
solution was subjected to LCMS (FA05) analysis, a compound
(Compound 133) in which an amide bond was formed between the
N-terminal amino group of
H-g-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip
and the side-chain carboxylic acid of Asp, which is a target
molecule, a N- to O-acyl shift product of the target molecule, a
product in which one of the hydroxyl groups of the target molecule
was esterified with TFA, a product in which two of the hydroxyl
groups of the target molecule were esterified with TFA, and a
product in which one of the hydroxyl groups of the N- to O-acyl
shift product of the target molecule was esterified with TFA were
confirmed to have a UV area ratio of 30:34:24:3:9. The LC analysis
results are shown in FIG. 20.
[0681] The data of FIG. 20 are shown below: [0682] Desired peptide
(Compound 133, compound in which an amide bond was formed between
the N-terminal amino group of
H-g-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip
and the side-chain carboxylic acid of Asp) [0683] LCMS (ESI)
m/z=1473.8 (M+H)+ [0684] Retention time: 0.78 minutes (analysis
condition SQDFA05) [0685] N- to O-acyl shift product of the target
molecule (a compound in which N- to O-acyl shift progressed at
either one or both of the two hydroxyl groups of the target
molecule) [0686] LCMS (ESI) m/z=1473.8 (M+H)+ [0687] Retention
time: 0.65 minutes (analysis condition SQDFA05) [0688] Compound in
which one of the hydroxyl groups of the target molecule was
esterified with TFA [0689] LCMS (ESI) m/z=1569.8 (M+H)+ [0690]
Retention time: 0.89 minutes (analysis condition SQDFA05) [0691]
Compound in which two of the hydroxyl groups of the target molecule
were esterified with TFA [0692] LCMS (ESI) m/z=1665.7 (M+H)+ [0693]
Retention time: 0.99 minutes (analysis condition SQDFA05) [0694]
Compound in which one of the hydroxyl groups of the N- to O-acyl
shift product of the target molecule was esterified with TFA [0695]
LCMS (ESI) m/z=1569.8 (M+H)+ [0696] Retention time: 0.73 minutes
(analysis condition SQDFA05)
[0697] According to the above-mentioned results, the problems of N-
to O-acyl shift and the problems of TFA esterification of the
hydroxyl groups included in the compounds could not be completely
solved simply by decreasing the reaction temperature and by using a
lower concentration of the TFA solution.
Example 6
Application to Parallel Synthesis (Solid Phase Method) of the
Present Invention
Example 6-1
Synthesis of a Group of Peptides in which Cyclization by an Amide
Bond was Performed Between the N-Terminal Amino Group and the
Side-Chain Carboxylic Acid Group of Aspartic Acid
[0698] A group of peptides in which cyclization by an amide bond
was performed between the N-terminal main chain amino group and the
side-chain carboxylic acid group of an aspartic acid to which
proline is bonded to its C terminus or its C terminus is amidated
(amidated by any one of piperidine, 4-(tert-butyl)piperidine, and
N-methyloctan-1-amine) was synthesized.
[0699] Using 100 mg of any one of Compound. 50 (2-chlorotrityl
resin made to support Compound 48 (Fmoc-Asp-pip)), Compound 52
(2-chlorotrityl resin made to support Compound 51
(Fmoc-Asp-piptBu)), Compound 55 (2-chlorotrityl resin made to
support Compound 54 (Fmoc-Asp-MeOctyl)), and Compound 58
(2-chlorotrityl resin made to support Compound 57
(Fmoc-Asp-Pro-OPis)), peptide elongation was carried out using as
an Fmoc amino acid Fmoc-MeVal-OH, Fmoc-MePhe(3-Cl)-OH (Compound
15), Fmoc-MePhe(4-Cl)-OH (Compound 16), Fmoc-MeHis(Trt)-OH
(Compound 7), Fmoc-MePhe-OH, Fmoc-MeSer(DMT)-OH (Compound 5),
Fmoc-MeSer(THP)-OH (Compound 6), Fmoc-MeAla-OH, Fmoc-nPrGly-OH
(Compound 20), Fmoc-MeGly-OH, Fmoc-Hyp(Et)-OH (Compound 18),
Fmoc-Pro-OH, Fmoc-Thr(THP)-OH (Compound 2), Fmoc-Ile-OH,
Fmoc-Val-OH, Fmoc-Trp-OH, Fmoc-Tyr(3-F,tBu)-OH (Compound 13),
Fmoc-Phe(4-CF3)-OH, Fmoc-Phe(3-Cl)-OH, Fmoc-Ser(Trt)-OH,
Fmoc-Met(O2)-OH, Fmoc-b-Ala-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, and such.
(In MeSer elongation, Fmoc-MeSer(DMT)-OH (Compound 5) was used for
PS-53 and PS-54 (Table 5-1), and Fmoc-MeSer(THP)-OH (Compound 6)
was used for the other cases.) Peptide elongation was performed
according to the peptide synthesis method by the Fmoc method
already described in the Examples. After peptide elongation, the
N-terminal Fmoc group was removed on the peptide synthesizer, and
then the resin was washed with DMF.
[0700] Then, after swelling the resin again with DCM, TFE/DCM (1/1,
v/v, 2 mL) and diisopropylethylamine (DIPEA, at an amount of 1.8
equivalents with respect to the number of moles on the resin used
(number of moles resulting from multiplying the loading rate
(mmol/g) by the amount of resin used (normally 0.10 g))) were added
to the resin, this was shaken at room temperature for two hours,
and then the peptides were cleaved off from the resin. Next, the
resin was removed by filtering the solution inside the tube through
a column for synthesis, and the remaining resin was further washed
twice with TFE/DCM (1/1, v/v, 1 mL). All of the obtained cleavage
solutions were mixed, and concentrated under reduced pressure.
[0701] The obtained residue was dissolved in DMF/DCM (1/1, v/v, 8
mL), a 0.5 M O-(7-aza-1H-benzotriazol
-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HATU)/DMF
solution (at an amount of 1.5 equivalents with respect to the
number of moles on the resin used (number of moles resulting from
multiplying the loading rate (mmol/g) of the resin by the amount of
resin used (0.1 g))) and DIPEA (1.8 equivalents with respect to the
number of moles on the resin used) were added, and this was stirred
at room temperature for two hours. Thereafter, the solvent was
distilled off under reduced pressure.
[0702] Deprotection was performed as follows on the obtained
residue.
[0703] When the sequence includes Tyr(3-F,tBu), 2 mL of 0.1 M
tetramethylammonium hydrogen sulfate/HFIP solution (2% TIPS)
prepared by the already described method was added to dissolve the
residue, and then this was left to stand at room temperature or at
30.degree. C. for 24 hours. When the sequence does not contain
Tyr(3-F), 4 mL of 0.05 M tetramethylammonium hydrogen sulfate/HFIP
solution (2% TIPS) prepared by the already described method was
added to dissolve the residue, and then this was left to stand at
room temperature for four hours. After allowing the reaction to
stand for a certain amount of time, diisopropylethylamine (DIPEA,
70 .mu.L) was added, and the solvent was distilled off under
reduced pressure.
[0704] After distilling off the solvent under reduced pressure, the
resulting material was dissolved in DMF. After removing the
insoluble material by filtration, this was purified by preparative
HPLC, and the amide-cyclized cyclic peptides in the title (PS-1 to
PS-54) were obtained. The sequences of PS-1 to PS-54 are shown in
Table 5-1 and their structures are shown in 5-2. The mass spectral
values, liquid chromatography retention times, purities, and yields
of each of the obtained peptides are shown in Table 5-3.
TABLE-US-00007 TABLE 5-1 Resin Loading MW 11 10 9 8 7 6 5 4 3 2 1
H-1 Used resin Amunt PS-1 1683.8 Ala Pro Met(O2) Tyr(3-F) MeHis Thr
Thr Trp MePhe(4-Cl) MePhe(4-Cl) Asp MeOctyl Compound 0.368 mmol/g
PS-2 1691.8 Ala Thr Met(O2) MePhe Thr MePhe(4-Cl) Phe(3-Cl)
MePhe(4-Cl) Thr Phe(3-Cl) Asp piptBu Compound 0.356 mmol/g PS-3
1722.8 Ala Thr Met(O2) MePhe Thr Trp Phe(3-Cl) MePhe Tyr(3-F)
Phe(3-Cl) Asp piptBu Compound 0.358 mmol/g PS-4 1652.2 Ala Thr
Phe(3-Cl) MeAla Met(O2) Trp Phe(3-Cl) MePhe Thr Phe(3-Cl) Asp
piptBu Compound 0.356 mmol/g PS-5 1699.8 Ser Pro Met(O2) Tyr(3-F)
MeHis Thr Thr Trp MePhe(4-Cl) MePhe(4-Cl) Asp MeOctyl Compound
0.368 mmol/g PS-6 1707.8 Ser Thr Met(O2) MePhe Thr MePhe(4-Cl)
Phe(3-Cl) MePhe(4-Cl) Thr Phe(3-Cl) Asp piptBu Compound 0.356
mmol/g PS-7 1745.8 Ser Thr Met(O2) MePhe Thr Trp Phe(3-Cl) MePhe
Tyr(3-F) Phe(3-Cl) Asp Meoctyl Compound 0.368 mmol/g PS-8 1655.8
Ala Pro Met(O2) Tyr(3-F) MeHis Thr Thr Trp MePhe(4-Cl) MePhe(4-Cl)
Asp Pro Compound 0.3885 mmol/g PS-9 1665.3 Ala Thr Met(O2) MePhe
Thr MePhe(4-Cl) Phe(3-Cl) MePhe(4-Cl) Thr Phe(3-Cl) Asp Pro
Compound 0.3885 mmol/g PS-10 1701.7 Ala Thr Met(O2) MePhe Thr Trp
Phe(3-Cl) MePhe Tyr(3-F) Phe(3-Cl) Asp Pro Compound 0.3885 mmol/g
PS-11 1626.1 Ala Thr Phe(3-Cl) MeAla Met(O2) Trp Phe(3-Cl) MePhe
Thr Phe(3-Cl) Asp Pro Compound 0.3885 mmol/g PS-12 1671.8 Ser Pro
Met(O2) Tyr(3-F) MeHis Thr Thr Trp MePhe(4-Cl) MePhe(4-Cl) Asp Pro
Compound 0.3885 mmol/g PS-13 1681.8 Ser Thr Met(O2) MePhe Thr
MePhe(3-Cl) Phe(3-Cl) MePhe(4-Cl) Thr Phe(3-Cl) Asp Pro Compound
0.3885 mmol/g PS-14 1717.7 Ser Thr Met(O2) MePhe Thr Trp Phe(3-Cl)
MePhe Tyr(3-F) Phe(3-Cl) Asp Pro Compound 0.3885 mmol/g PS-15
1642.1 Ser Thr Phe(3-Cl) MeAla Met(O2) Trp Phe(3-Cl) MePhe Thr
Phe(3-Cl) Asp Pro Compound 0.3885 mmol/g PS-16 1543.09 Ala
MePhe(O2) MePhe(3-Cl) Met(O2) Hyp(Et) Th MePhe(3-Cl) nPrGly bAla
MeGly Asp piptBu Compound 0.356 mmol/g PS-17 1690.3 Ala Met(O2)
MePhe Trp Pro MeGly MePhe(4-Cl) Met(O2) MeVal Trp Asp pip Compound
0.338 mmol/g PS-18 1808.4 Ala Met(O2) Met(O2) MePhe Met(O2) Trp
Phe(3-Cl) MePhe(4-Cl) Thr Phe(3-Cl) Asp MeOctyl Compound 0.366
mmol/g PS-19 1664.23 Ala Ile Phe(9-Cl) MeAla Thr Trp Phe(3-Cl)
MePhe Met(O2) Phe(3-Cl) Asp piptBu Compound 0.336 mmol/g PS-20
1561.11 Ser MePhe(3-Cl) MePhe(3-Cl) Met(O2) Hyp(Et) Thr MePhe(4-Cl)
nPrGly bAla MeGly Asp MeOctyl Compound 0.366 mmol/g PS-21 1864.43
Ser Met(O2) MePhe Trp Pro MeGly MePhe(3-Cl) Met(O2) MeVal Trp Asp
MeOctyl Compound 0.368 mmol/g PS-22 1824.4 Ser Met(O2) Met(O2)
MePhe Met(O2) Trp Phe(3-Cl) MePhe(4-Cl) Thr Phe(3-Cl) Asp MeOctyl
Compound 0.388 mmol/g PS-23 1680.23 Ser Ile Phe(3-Cl) MeAla Thr Trp
Phe(3-Cl) MePhe Mer(O2) Phe(3-Cl) Asp piptBu Compound 0.358 mmol/g
PS-24 1518.97 Ala MePhe(3-Cl) MePhe(3-Cl) Met(O2) Hyp(Et) Thr
MePhe(3-Cl) nPrGly bAla MnGly Asp Pro Compound 0.3885 mmol/g PS-25
1620.29 Ala Met(O2) MePhe Trp Pro MeGly MePhe(3-Cl) Met(O2) MeVal
Trs Asp Pro Compound 0.3885 mmol/g PS-26 1780.25 Ala Met(O2)
Met(O2) MePhe Met(O2) Trp Phe(3-Cl) MePhe(4-Cl) Thr Phe(3-Cl) Asp
Pro Compound 0.3885 mmol/g PS-27 1838.11 Ala Ile Phe(3-Cl) MeAla
Thr Trp Phe(3-Cl) MePhe Met(O2) Phe(3-Cl) Asp Pro Compound 0.3885
mmol/g PS-28 1532.97 Ser MePhe(3-Cl) MePhe(3-Cl) Met(O2) Hyp(Et)
Thr MePhe(3-Cl) MePhe(4-Cl) Thr MeGly Asp Pro Compound 0.3885
mmol/g PS-29 1636.29 Ser Met(O2) MePhe Trp Pro MeGly MePhe(3-Cl)
MePhe(4-Cl) Thr Trp Asp Pro Compound 0.3885 mmol/g PS-30 1796.25
Ser Met(O2) Met(O2) MePhe Met(O2) Trp Phe(3-Cl) MePhe(4-Cl) Thr
Phe(3-Cl) Asp Pro Compound 0.3885 mmol/g PS-31 1654.11 Ser Ile
Phe(3-Cl) MeAla Thr Trp Phe(3-Cl) MePhe Thr Phe(3-Cl) Asp Pro
Compound 0.3885 mmol/g PS-32 1575.57 Ala Thr Met(O2) MePhe Thr Trp
Phe(3-Cl) MePhe(4-Cl) MeSer MeSer Asp Pro Compound 0.3885 mmol/g
PS-33 1678.8 Ala Met(O2) Phe(3-Cl) MeSer Thr Trp Phe(3-Cl)
MePhe(4-Cl) Thr Phe(3-Cl) Asp Pro Compound 0.3885 mmol/g PS-34
1808.04 Ala Thr Met(O2) Ile Thr Trp Phe(3-Cl) MePhe(4-Cl) Thr
Phe(3-Cl) Asp Pro Compound 0.3885 mmol/g PS-35 1513.95 Ala Ile Gly
Ile Thr Trp Phe(3-Cl) MePhe(4-Cl) Thr Phe(3-Cl) Asp Pro Compound
0.3885 mmol/g PS-36 1379.94 Ser bAla Met(O2) Gly MePhe(3-Cl) Pro
bAla Mer(O2) Thr MePhe Asp Pro Compound 0.3885 mmol/g PS-37 1591.87
Ser Thr Met(O2) MePhe Thr Trp Phe(3-Cl) MePhe(4-Cl) MeSer MeSer Asp
Pro Compound 0.3885 mmol/g PS-38 1682.5 Ser Met(O2) Phe(3-Cl) MeSer
Thr Trp Phe(3-Cl) MePhe(4-Cl) Thr Phe(3-Cl) Asp Pro Compound 0.3885
mmol/g PS-39 1624.04 Ser Thr Met(O2) Ile Thr Trp Phe(3-Cl)
MePhe(4-Cl) Thr Phe(3-Cl) Asp Pro Compound 0.3885 mmol/g PS-40
1529.9 Ser Ile Gly Ile Thr Trp Phe(3-Cl) MePhe(4-Cl) Thr Phe(3-Cl)
Asp Pro Compound 0.3885 mmol/g PS-41 1382.08 Ala bAla Met(O2) Gly
MePhe(3-Cl) Pro bAla MePhe(4-Cl) Thr MePhe Asp MeOctyl Compound
0.366 mmol/g PS-42 1693.7 Ala Thr Met(O2) MePhe Thr Trp Phe(3-Cl)
MePhe(4-Cl) MeSer MeSer Asp MeOctyl Compound 0.366 mmol/g PS-43
1702.62 Ala Met(O2) Phe(3-Cl) MeSer Thr Trp Phe(3-Cl) MePhe(4-Cl)
Thr Phe(3-Cl) Asp piptBu Compound 0.361 mmol/g PS-44 1619.7 Ser Thr
Mat(O2) MePhe Thr Trp Phe(3-Cl) MePhe(4-Cl) Thr MeSer Asp MeOctyl
Compound 0.366 mmol/g PS-45 1718.62 Ser Met(O2) Phe(3-Cl) MeSer Thr
Trp Phe(3-Cl) MePhe(4-Cl) Thr Phe(3-Cl) Asp piptBu Compound 0.361
mmol/g PS-46 1650.16 Ser Met(O2) MeSer MeVal Thr Trp Phe(3-Cl)
MePhe(4-Cl) Thr Phe(3-Cl) Asp piptBu Compound 0.361 mmol/g PS-47
1634.16 Ala Thr Met(O2) Ile Thr Trp Phe(3-Cl) MePhe(4-Cl) Thr
Phe(3-Cl) Asp piptBu Compound 0.361 mmol/g PS-48 1483.97 Ala Ile
Gly Ile Thr Trp Phe(3-Cl) MePhe(4-Cl) Thr Phe(3-Cl) Asp Pip
Compound 0.320 mmol/g PS-49 1650.16 Ser Thr Met(O2) Ile Thr Trp
Phe(3-Cl) MePhe(4-Cl) Thr Phe(3-Cl) Asp piptBu Compound 0.361
mmol/g PS-50 1558.07 Ser Ile Gly Ile Thr Trp Phe(3-Cl) MePhe(4-Cl)
Thr Phe(3-Cl) Asp piptBu Compound 0.361 mmol/g PS-51 1397.47 Ala
Pro bAla Phe(4-CF3) Thr Ser bAla Try Trp Val Asp Pro Compound
0.3573 mmol/g PS-52 1579.00 Ala Phe(3-Cl) MePhe(4-Cl) MeHis MeGly
Phe(3-Cl) MePhe Thr MeHis Thr Asp Pro Compound 0.3573 mmol/g PS-53
1789.9 Ala Met(O2) Phe(3-Cl) MeSer Met(O2) Trp MePhe MePhe Tyr(3-F)
Phe(3-Cl) Asp piptBu Compound 0.361 mmol/g PS-54 1763.8 Ala Met(O2)
Phe(3-Cl) MeSer Met(O2) Trp MePhe MePhe Tyr(3-F) Phe(3-Cl) Asp Pro
Compound 0.375 mmol/g indicates data missing or illegible when
filed
TABLE-US-00008 TABLE 5-2 PS-1 ##STR00080## PS-2 ##STR00081## PS-3
##STR00082## PS-4 ##STR00083## PS-5 ##STR00084## PS-6 ##STR00085##
PS-7 ##STR00086## PS-8 ##STR00087## PS-9 ##STR00088## PS-10
##STR00089## PS-11 ##STR00090## PS-12 ##STR00091## PS-13
##STR00092## PS-14 ##STR00093## PS-15 ##STR00094## PS-16
##STR00095## PS-17 ##STR00096## PS-18 ##STR00097## PS-19
##STR00098## PS-20 ##STR00099## PS-21 ##STR00100## PS-22
##STR00101## PS-23 ##STR00102## PS-24 ##STR00103## PS-25
##STR00104## PS-26 ##STR00105## PS-27 ##STR00106## PS-28
##STR00107## PS-29 ##STR00108## PS-30 ##STR00109## PS-31
##STR00110## PS-32 ##STR00111## PS-33 ##STR00112## PS-34
##STR00113## PS-35 ##STR00114## PS-36 ##STR00115## PS-37
##STR00116## PS-38 ##STR00117## PS-39 ##STR00118## PS-40
##STR00119## PS-41 ##STR00120## PS-42 ##STR00121## PS-43
##STR00122## PS-44 ##STR00123## PS-45 ##STR00124## PS-46
##STR00125## PS-47 ##STR00126## PS-48 ##STR00127## PS-49
##STR00128## PS-50 ##STR00129## PS-51 ##STR00130## PS-52
##STR00131## PS-53 ##STR00132## PS-54 ##STR00133##
TABLE-US-00009 TABLE 5-3 Reten- tion LCMS LCMS Time (ESI) Purity
Yield Condition (min) m/z (%) (mg) PS-1 SQDFA05 0.75 1682.8 (M +
H)+ 85 4.81 PS-2 SQDFA05 1.09 1689.7 (M + H)+ 95 5.15 PS-3 SQDFA05
0.98 1726.7 (M + H)+ 90 8.08 PS-4 SQDFA05 1.04 1650.8 (M + H)+ 95
13.82 PS-5 SQDFA05 0.74 1698.8 (M + H)+ 95 4.9 PS-6 SQDFA05 1.08
1705.9 (M + H)+ 95 10.61 PS-7 SQDFA05 1.02 1744.7 (M + H)+ 95 6.53
PS-8 SQDFA05 6.57 1654.7 (M + H)+ 95 8.86 PS-9 SQDFA05 0.93 1663.5
(M + H)+ 95 9.09 PS-10 SQDFA05 0.84 1700.7 (M + H)+ 90 10.5 PS-11
SQDFA05 0.89 1624.7 (M + H)+ 95 11.32 PS-12 SQDFA05 0.58 1670.7 (M
+ H)+ 95 3.32 PS-13 SQDFA05 0.92 1679.6 (M + H)+ 95 8.71 PS-14
SQDFA05 0.83 1716.7 (M + H)+ 95 7.4 PS-15 SQDFA05 0.88 1640.7 (M +
H)+ 95 7.9 PS-16 SQDFA05 0.99 1541.9 (M + H)+ 70 4.94 PS-17 SQDFA05
0.81 1589.9 (M + H)+ 85 11.3 PS-18 SQDFA05 1.06 1806.8 (M + H)+ 90
11.94 PS-19 SQDFA05 1.08 1662.8 (M + H)+ 90 13.25 PS-20 SQDFA05
1.02 1559.7 (M + H)+ 60 1.77 PS-21 SQDFA05 0.93 1663.9 (M + H)+ 90
4.33 PS-22 SQDFA05 1.05 1822.8 (M + H)+ 90 11.56 PS-23 SQDFA05 1.07
1678.8 (M + H)+ 95 6.02 PS-24 SQDFA05 0.79 1515.7 (M + H)+ 75 3.52
PS-25 SQDFA05 0.76 1619.9 (M + H)+ 70 8.35 PS-26 SQDFA05 0.88
1778.5 (M + H)+ 80 11.88 PS-27 SQDFA05 0.91 1636.8 (M + H)+ 80
13.93 PS-28 SQDFA05 0.77 1531.7 (M + H)+ 70 6.43 PS-29 SQDFA05 0.73
1635.7 (M + H)+ 91 3.78 PS-30 SQDFA05 0.87 1794.7 (M + H)+ 95 11.86
PS-31 SQDFA05 0.91 1652.7 (M + H)+ 83 10.33 PS-32 SGDFA05 0.74
1574.7 (M + H)+ 95 13.75 PS-33 SQDFA05 0.86 1674.6 (M + H)+ 94
15.42 PS-34 SQDFA05 0.84 1606.6 (M + H)+ 95 12.53 PS-35 SGDFA05
0.89 1512.7 (M + H)+ 95 16.18 PS-36 SQDFA05 0.53 1379.7 (M + H)+ 80
2.12 PS-37 SQDFA05 0.73 1590.7 (M + H)+ 95 15.65 PS-38 SQDFA05 0.85
1690.4 (M + H)+ 95 11.25 PS-39 SQDFA05 0.82 1622.6 (M + H)+ 95
11.33 PS-40 SQDFA05 0.87 1528.7 (M + H)+ 94 11.61 PS-41 SQDFA05
0.73 1391.7 (M + H)+ 90 2.32 PS-42 SQDFA05 0.92 1602.7 (M + H)+ 90
6.54 PS-43 SQDFA05 1.02 1700.6 (M + H)+ 90 5.83 PS-44 SQDFA05 0.91
1618.9 (M + H)+ 90 4.85 PS-45 SQDFA05 1.00 1716.7 (M + H)+ 90 3.93
PS-46 SQDFA05 0.98 1648.7 (M + H)+ 90 4.77 PS-47 SQDFA05 0.99
1632.7 (M + H)+ 90 3.64 PS-48 SQDFA05 0.96 1482.7 (M + H)+ 90 4.81
PS-49 SQDFA05 0.97 1648.7 (M + H)+ 90 3.15 PS-50 SQDFA05 1.05
1554.8 (M + H)+ 90 6.01 PS-51 SQDFA05 0.62 1396.7 (M + H)+ 71 2.98
PS-52 SQDFA05 0.56 1577.6 (M + H)+ 95 6.4 PS-53 SQDFA05 0.93 1788.7
(M + H)+ 89 7.4 PS-54 SQDFA05 0.78 1762.7 (M + H)+ 85 2.8
Example 7
Application of the Present Invention to the Liquid Phase Method
[0705] Synthesis Including Elongation Reaction by the Liquid Phase
Method is Shown Below
Partial Application to Liquid Phase Methods
Synthesis of the Cyclic Peptide (Compound 154, Cyclic Peptide in
which an Amide Bond was Formed Between the Main Chain N-Terminal
Amino Group of
H-Ala-Trp-Nle-Trp-Ser-nPrGly-MePhe(3-Cl)-MeHis-MeGly-Pro-Asp-Pro-OH
and the Side-Chain Carboxylic Acid Group of Asp), which Includes
Coupling of Fmoc-Ala-OSu (Compound 152) with
H-Trp-Nle-Trp-Ser(THP)-nPrGly-MePhe(3-Cl)-MeHis(Trt)-MeGly-Pro-Asp-Pro-OP-
is (Compound 151) in the Liquid Phase
[0706] The peptide was synthesized by the synthetic route described
in FIG. 21 which includes elongation reaction by the liquid phase
method.
Example 7-1
Synthesis of
H-Trp-Nle-Trp-Ser(THP)-nPrGly-MePhe(3-Cl)-MeHis(Trt)-MeGly-Pro-Asp-Pro-OP-
is (Compound 151)
##STR00134##
[0708] Peptide elongation was performed according to the peptide
synthesis method by the Fmoc method already described in the
Examples, using Fmoc-Asp(O-Trt(2-Cl)-resin)-Pro-OPis (Compound 58,
loading rate: 0.3736 mmol/g, 200 mg) synthesized by the already
described method. After peptide elongation, removal of the
N-terminal Fmoc group was performed on the peptide synthesizer, and
the resin was washed using DMF.
[0709] Then, after swelling the resin again with DCM, TFE/DCM (1/1,
v/v, 4 mL) and diisopropylethylamine (24 .mu.L) were added to the
resin, this was shaken at room temperature for two hours, and the
peptide was cleaved off from the resin. Next, the resin was removed
by filtering the solution inside the tube through a column for
synthesis, and the remaining resin was further washed twice with
TFE/DCM (1/1, v/v, 2 mL). All of the obtained cleavage solutions
were mixed, concentrated under reduced pressure, and the titled
compound (Compound 151,
H-Trp-Nle-Trp-Ser(THP)-nPrGly-MePhe(3-Cl)-MeHis(Trt)-MeGly-Pro-Asp-Pro-OP-
is; 113.8 mg) was obtained. This was used in the next step without
purification. [0710] LCMS (ESI) m/z=1860.9 (M+H).sup.+ [0711]
Retention time: 0.72 minutes (analysis condition SQDFA05)
Example 7-2
Synthesis of (S)-2,5-dioxopyrrolidin-1-yl
2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoate (Compound
152, Fmoc-Ala-OSu)
##STR00135##
[0713] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic
acid (Fmoc-Ala-OH; 1.00 g, 3.21 mmol),
1-hydroxypyrrolidin-2,5-dione (HOSu; 0.554 g, 4.82 mmol), and
dichloromethane (6.4 mL) were mixed under nitrogen atmosphere. This
mixture was cooled on ice to 0.degree. C.,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(WSCIHCl, 0.924 g, 4.82 mmol) was added, and the obtained reaction
solution was stirred at 0.degree. C. for one hour and at room
temperature for 15 hours. Next, the solvent was distilled off under
reduced pressure, and the obtained residue was purified by
reverse-phase silica gel chromatography (0.1% aqueous formic acid
solution/0.1% formic acid solution in acetonitrile) obtain
(S)-2,5-dioxopyrrolidin-1-yl
2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoate (Compound
152, Fmoc-Ala-OSu, 1.05 g) [0714] LCMS (ESI) m/z=409.3 (M+H).sup.+
[0715] Retention time: 0.80 minutes (analysis condition
SQDFA05)
Example 7-3
Coupling Between (S)-2,5-dioxopyrrolidin-1-yl
2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoate (Compound
152, Fmoc-Ala-OSu) and
H-Trp-Nle-Trp-Ser(THP)-nPrGly-MePhe(3-Cl)-MeHis(Trt)-MeGly-Pro-Asp-Pro-OP-
is (Compound 151), and the Subsequent Fmoc Removal Reaction
##STR00136##
[0717] (S)-2,5-Dioxopyrrolidin-1-yl
2-((((9H-fluoren-9-yl)methoxy)carbonyl)-amino)propanoate (Compound
152, Fmoc-Ala-OSu, 26.2 mg) and diisopropylethylamine (DIPEA, 12.8
.mu.L) were added to a solution of the obtained
H-Trp-Nle-Trp-Ser(THP)-nPrGly-MePhe(3-Cl)-MeHis(Trt)-MeGly-Pro-Asp-Pro-OP-
is (Compound 151, 113.8 mg) in dichloromethane (245 .mu.L), and
this was stirred at 25.degree. C. for one hour. Next, methylamine
(40% solution in methanol, 11.9 .mu.L) was added to the reaction
solution, this was stirred for 30 minutes. Then, DBU (11.1 .mu.L)
was added, and this was stirred for another 30 minutes. The
obtained reaction solution was purified by reverse-phase silica gel
column chromatography (0.1% aqueous formic acid solution/0.1%
formic acid solution in acetonitrile), and the obtained fractions
were freeze-dried. The obtained residue was dissolved in
dichloromethane, and this washed with saturated aqueous sodium
hydrogen carbonate solution and saturated aqueous sodium chloride
solution to obtain
H-Ala-Trp-Nle-Trp-Ser(THP)-nPrGly-MePhe(3-Cl)-MeHis(Trt)-MeGly-Pro-Asp-Pr-
o-OPis (Compound 153; 79.3 mg, 0.041 mmol). [0718] LCMS (ESI)
m/z=1931.8 (M+H).sup.+ [0719] Retention time: 0.73 minutes
(analysis condition SQDFA05)
Example 7-4
Synthesis of the Compound in which Amide Cyclization is
Accomplished Between the N-Terminal Amino Group of
H-Ala-Trp-Nle-Trp-Ser-nPrGly-MePhe(3-Cl)-MeHis-MeGly-Pro-Asp-Pro-OH
and the Side-Chain Carboxylic Acid Group of Asp (Compound 154)
(Cyclization Reaction of Compound 153, and the Subsequent
Deprotection Reaction)
##STR00137##
[0721] The obtained
H-Ala-Trp-Nle-Trp-Ser(THP)-nPrGly-MePhe(3-Cl)-MeHis(Trt)-MeGly-Pro-Asp-Pr-
o-OPis (Compound 153; 79.3 mg, 0.041 mmol) was dissolved in DMF (20
mL) and dichloromethane (20 mL), HATU (17.2 mg, 0.045 mmol) and
diisopropylethylamine (10.8 .mu.L, 0.062 mmol) were added, and this
was stirred at 25.degree. C. for two hours. Subsequently, the
solvent was distilled off under reduced pressure. Then, 0.05 M
tetramethylammonium hydrogen sulfate/HFIP (2% TIPS) solution
(prepared by the method already described in the Examples, 8 mL)
was added and this was left to stand at 25.degree. C. for one hour.
Diisopropylethylamine (140 .mu.L) was added to the obtained
reaction solution and the solvent was distilled off under reduced
pressure. The obtained residue was purified by reverse-phase silica
gel column chromatography (0.1% aqueous formic acid solution/0.1%
formic acid solution in acetonitrile), and the obtained fractions
were freeze-dried to obtain the compound in which amide cyclization
is accomplished between the N-terminal amino group of
H-Ala-Trp-Nle-Trp-Ser-nPrGly-MePhe(3-Cl)-MeHis-MeGly-Pro-Asp-Pro-OH
and the side-chain carboxylic acid group of Asp (Compound 154; 59
mg, 0.040 mmol, 98%).
[0722] LCMS (ESI) m/z=1469.7 (M+H)+
[0723] Retention time: 0.61 minutes (analysis condition
SQDFA05)
[0724] As in the above-described peptide syntheses which include
segment coupling in the liquid phase, the synthesis methods of the
present invention can be applied to liquid-phase methods as
well.
INDUSTRIAL APPLICABILITY
[0725] According to the present invention, peptides containing
N-substituted amino acids, which are expected to be useful as
pharmaceuticals, can be synthesized with high purity and high
synthetic efficiency. The present invention is useful in fields
such as industrial production of peptides comprising N-substituted
amino acids, which may become raw materials for
pharmaceuticals.
* * * * *